Fluorescent protein sensors of post-translational modifications

ABSTRACT

The present invention includes a fluorescent compound that can detect an activity. such as an enzymatic activity, and exhibits quenching. The fluorescent compound can include a fluorescent protein, such as an Aequorea-related green fluorescent protein. The fluorescent compound can include a substrate site for an enzymatic activity such as a kinase activity, a phosphatase activity, a protease activity, and a glycosylase activity The fluorescent compound of the present invention can be used to detect such enzymatic activities in samples, such as biological samples, including cells. The present invention also includes nucleic acids that encode the fluorescent compounds of the present inventions, and cells that include such nucleic acids or fluorescent compounds.

TECHNICAL FIELD

[0001] The present invention generally relates to compositions andmethods for the detection of activities, such as enzymatic activities,using fluorescent compounds that are modified by the activity such thatthey exhibit a change in their sensitivity to quenching.

BACKGROUND

[0002] Fluorescent compounds have been used in the art to detect a widevariety of biological phenomenon, such as changes in ion concentration,specific binding reactions. subcellular localization, and enzymaticreactions. In the case of detecting changes in ion concentration,specific binding reactions, and subcellular localization, thefluorescent compound is used as a label to detect such specific bindingor localization. In some cases, the fluorescence of the fluorescentcompound is altered after an enzyme has acted on the fluorescentcompound or a molecule binds with the fluorescent compound. For example,the activity of beta-galactosidase on the substrate fluoresceindi-beta-D-galactopyranoside causes an increase in fluorescence of thesubstrate (Molecular Probes Catalogue, Sixth Edition, p. 208 (1996)).Likewise, the action of beta-lactamase on CCF2-AM causes the compound tochange fluorescence from green to blue due to an uncoupling offluorescence resonance energy transfer (FPET) (Tsien and Zlokamik, WO96/30540, published Oct. 3, 1996). Protease activity can also bedetected by the action of a protease on a fluorescent compound thatuncouples FRET of the fluorescent compound (Tsien et al., WO 97/28261,published Aug. 7, 1997).

[0003] The detection of enzymatic reactions is important for the studyof biological phenomenon, cellular biology, medical diagnostics, anddrug discovery. Several classes of enzymes have been implicated indisease states, such as proteases for HIV and kinases for cancer. Drugdiscovery preferably uses living cells to detect compounds that canalter the activity enzymes involved in such disease states. However, exvivo methods can also be used in drug discovery

[0004] Protein kinases and phosphatases have particularly beenrecognized as one of the more important general mechanism of cellularregulation. Protein phosphorylation commonly occurs on three major aminoacids, tyrosine, serine or threonine. Changes in the phosphorylationstate of these amino acids within proteins can regulate many aspects ofcellular metabolism, regulation, grown and differentiation. Changes inthe phosphorylation state of proteins, mediated through phosphorylationby kinases, or dephosphoryation by phosphatases, is a common mechanismthrough which cell surface signaling pathways transmit and integrateinformation into the nucleus. Given their key role in cellularregulation, it is not surprising that defects in protein kinases andphosphatases have been implicated in many disease states and conditions.For example, the over-expression of cellular tyrosine kinases such asthe EGF or PDGF receptors, or the mutation of tyrosine kinases toproduce constitutively active forms (oncogenes) occurs in many cancercells (Durker et al. Nature Medicine 2:561-556 (1996)). Protein tyrosinekinases are also implicated in inflammatory signals, and defectiveThr/Ser kinase genes have been demonstrated to be implicated in severaldiseases such as myotonic dystrophy, cancer and Alzheimer's disease(Sanpei et al., Biochem. Biophys. Res. Commun. 212:341-346 (1995);Sperger et al., Neurosci Lett. 197:149-153 (1995); Grammas et al.,Neurobiology of Aging, 16:563-569 (1995); Govani et al., Ann. N.Y. Acad.Sci. 777:332-337 (1996)).

[0005] The involvement of proteases, protein kinases, proteinphosphatases, and other classes of enzymes in disease states makes themattractive targets for the therapeutic intervention of drugs. In fact,many clinically useful drugs act on protein kinases or phosphatases.Examples include cyclosporin A, a potent immunosuppresent that binds tocyclophilin. This complex binds to the Ca⁺⁺/calmodulin-dependent proteinphosphatase type 2B (calcineurin), inhibiting its activity, and hencethe activation of T cells. Inhibitors of protein kinase C are inclinical trials as therapeutic agents for the treatment of cancer (Clin.Cancer Res. 1:113-122 (1995)) as are inhibitors of cyclin dependentkinase (J. Mol. Med. 73:509-514 (1995)).

[0006] The number of known enzymes, such as kinases and phosphatases,are growing rapidly as the influence of genomic programs to identify themolecular basis for diseases have increased in size and scope. Thesestudies are likely to implicate many more genes that encode enzymes thatare involved in the development and propagation of diseases in thefuture, thereby making them attractive targets for drug discovery.However, current methods of measuring enzyme activity, such as proteinphosphorylation and dephosphorylation, have many disadvantages whichprevents or limits the ability to rapidly screen for drugs usingminiaturized automated formats of many thousands of compounds. In thecase of phosphatases and kinases, this is because current methods relyon the incorporation and measurement of ³²P into the protein substratesof interest. In whole cells this necessitates the use of high levels ofradioactivity to efficiently label the cellular ATP pool and to ensurethat the target protein is efficiently labeled with radioactivity. Afterincubation with test drugs, the cells must be lysed and the protein ofinterest purified to determine its relative degree of phosphorylation.This method requires high numbers of cell, long preincubation times,careful manipulation, and washing steps to avoid artifactalphosphorylation or dephosphorylation. Furthermore, this approachrequires purification of the target protein, and final radioactiveincorporation into target proteins is usually very low, giving the assaypoor sensitivity. Alternative assay methods, such as those based onphosphorylation-specific antibodies using ELISA-type approaches, involvethe difficulty of producing antibodies that distinguish betweenphosphorylated and non-phosphorylated proteins, and the requirement forcell lysis, multiple incubations, and washing stages which are timeconsuming, complex to automate, and potentially susceptible toartifacts.

[0007] Fluorescent molecules are attractive as reporter molecules inmany assay systems because of their high sensitivity and ease ofquantification. Recently, fluorescent proteins have been the focus ofmuch attention because they can be produced in vivo by biologicalsystems and can be used to trace and monitor intracellular event withoutthe need to be introduced into the cell through microinjection orpermeabilization. The green fluorescent protein of Aequorea Victoria isparticularly interesting as a fluorescent indicator protein. A cDNA forthe protein has been cloned (Prasher et al., Gene 111:229-233 (1992)).Not only can the primary amino acid sequence of the protein be expressedfrom the cDNA, but the expressed protein can fluoresce in cells in vivo.

[0008] Fluorescent proteins have been used as markers of gene expressiontracers of cell lineage, and as fusion tags to monitor proteinlocalization within living cells (Rizzuto et al., Current Biol.6:183-188 (1996)); Cubitt et al., TIBS 20:448-455 (1995); U.S. Pat. No.5,625,048 to Tsien et al, issued Apr. 29, 1997). Furthermore, mutantversions of green fluorescent protein have been identified that exhibitaltered fluorescence characteristics, including altered excitation andemission maxima, as well as excitation and emission spectra of differentshapes. (Heim, Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994); Heim etal., Nature 373:663-665 (1995); U.S. Pat. No. 5,625,048, Tsien et al.,issued Apr. 29, 1997; WO 97/28261 to Tsien et al, published Aug. 7,1997; PCT/US 97/12400 to Tsien, filed Jul. 16, 1997; and PCT/US 97/14593by Tsien, filed Aug. 15, 1997). These proteins add variety and utilityto the arsenal of biologically based fluorescent indicators.

[0009] There is thus a need for assays for enzymes, such as thoseinvolved in protein phosphorylation, that are sensitive, simple to use,useful in living cells, and adaptable to high throughput screeningmethods. Preferably, such assays would not utilize radioactive materialsso that the assays would be safe and not generate hazardous wastes. Thepresent invention addresses these needs, and provides additionalbenefits as well.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1 depicts the nucleotide sequence (SEQ ID NO:1) and deducedamino acid sequence (SEQ ID NO:2) of wild-type Aequorea greenfluorescent protein.

[0011]FIG. 2 depicts a list of the positions and amino acid changes madefor phosphorylation mutants made more than fifteen amino acids in theprimary sequence from the N-terminus, as compared to FIG. 1. Amino acidsunderlined represent the phosphorylation motif, amino acids in bracketsrepresent wild type sequence at those positions.

[0012]FIG. 3 depicts the bacterial expression plasmid pRSET (Invitrogen)containing a region encoding GFP that is fused in frame with nucleotidesencoding an N-terminal polyhistidine tag.

[0013]FIG. 4 depicts a duel expression vector having expression controlsequences operably linked to sequences encoding for the expression ofprotein kinase A catalytic subunit (PKA cat) upstream from sequencescoding for the expression of a fluorescent protein substrate.

[0014]FIG. 5 depicts the effect of incubation time on the stability ofGFP mutant K8 fluorescence after termination of a kinase reaction with100 mM acetate buffer, 100 mM NaCl, 25 mM beta-glycerol phosphate pH5.0.

[0015]FIG. 6 depicts the effects of quenching on the fluorescentproperties of the GFP mutant either after phosphorylation by proteinkinase A or in the absence of protein kinase A.

[0016]FIG. 7 depicts the kinetics of phosphorylation of a GFP having akinase motif

[0017]FIG. 8 depicts the determination of the dose dependent inhibitionof PKA by known inhibitors of that enzyme using GFB mutant KS.

[0018]FIG. 9 depicts the phosphorylation of GFP with and without amembrane association motif

SUMMARY

[0019] The present invention recognizes that fluorescent compounds, suchas fluorescent proteins, can exist in at least two states and that thefluorescent properties of these two states can be different, preferablyafter exposure of the fluorescent compound to quenching conditions.Particularly, the stability of the fluorescence in one state can bedifferent from the stability of the fluorescence in the second state,which can be detected by their susceptibility to quenching. The firstand second states of the fluorescent compound can be caused by theaction of a chemical or enzyme. Thus, the present invention recognizesthat such fluorescent compounds can be used to detect various chemicalor enzymatic activities in a sample. The present invention recognizesthat fluorescent compounds can be fluorescent proteins that can beexpressed in cells. Thus, the fluorescent proteins can be used as invivo monitors of enzymatic activity (intracellular or extracellular) andcan be used to screen compounds for drugs that modulate (i.e., increaseor decrease the activity) an enzymatic activity. The present inventionalso provides nucleic acid molecules that encode fluorescent proteinsthat exhibit quenching.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Definitions

[0021] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this. invention belongs. Generally, thenomenclature used herein, and the laboratory procedures in cell culture,molecular genetics, and nucleic acid chemistry and hybridizationdescribed below, are those well known and commonly employed in the art.Standard techniques are used for recombinant nucleic acid methods,polynucleotide synthesis, and microbial culture and transformation(e.g., electroporation, and lipofection). Generally, enzymatic reactionsand purification steps are performed according to the manufacturer'sspecifications when kits or purchased reagents or materials are used.The techniques and procedures are generally performed according toconventional methods in the art and various general references (seegenerally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.) which are provided throughout this document. The nomenclature andlaboratory procedures used herein are those well known and commonlyemployed in the art. As used throughout the disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings.

[0022] “Quenching” or “quenching conditions,” as used herein, meansconditions that can cause a change in at least one fluorescent propertyof a fluorescent compound in a first state as compared to a secondstate. Quenching can be used, for example, to detect or measure thepresence or concentration of the fluorescent compound in the first stateor second state. Quenching conditions can include at least one quenchingagent.

[0023] A first state or a second state of a fluorescent compound meansthat the fluorescent compound exists in at lest two states, wherein thedifferent states have different fluorescent properties that can bedetected by quenching. The first state can differ from the second state,for example, by the covalent attachment of moieties to the fluorescentcompound, the binding or association of moieties to the fluorescentcompound, the cleavage or disruption of covalent or non-covalent bondsor interactions on or within the fluorescent compound, the binding orassociation of the fluorescent compound to other moieties or itself, ora change in the conformation of the fluorescent compound as a result ofthe presence of an activity.

[0024] “Quenching agent,” as used herein, can be any chemical, compoundor biological molecule that can cause quenching of a fluorescentcompound, either alone or in combination with other agents or factors

[0025] A “fluorescent compound,” as used herein, can be any fluorescentchemical or compound that can exhibit at least one different fluorescentproperty in a first state and a second state under quenching,conditions. For example, fluorescent compounds can be small aromaticcompounds such as fluorescein or rhodamine, or a weakly ornon-fluorescent compound such as, for example carbohydrates, lipids,proteins, peptides, polypeptides, nucleic acids that has been labeledwith a highly fluorescent compound or combinations thereof. For example,a fluorescent compound can be a fluorescent protein moiety. Afluorescent compound, such as those comprising a fluorescent proteinmoiety, can be soluble, membrane bound, or membrane associated. Membranebound versions of soluble fluorescent protein moieties can be made byadding, for example, hydrophobic regions such as signal sequences orhydrophobic moieties as they are known in the art using establishedmethods. Likewise, membrane associated versions of soluble fluorescentprotein moieties can be made by adding, for example, membraneassociation motifs, such as poly-Lys, to such fluorescent proteinmoieties using established methods.

[0026] A “fluorescent protein moiety” means any protein or fragmentthereof capable of fluorescence when excited with appropriateelectromagnetic radiation. This includes fluorescent proteins whoseamino acid sequences are either naturally occurring or engineered (i.e.,analogs) and proteins that have been modified to be fluorescent, such asby the addition of a fluorescence compound, such as fluorescein,rhodamine, Cy3-5, Cy-PE, lucifer yellow, C6-NBD, Dio-Cn(3), FITC,Biodipy-FL, eosin, propidium iodide, tetramethyl rhodamine B,Dil-Cn-(3), Lissamine Rhodamine B, Texas Red, Allophycocyanin, Dil-Cy-5,and squaranes by methods known in the art. For fluorescent compounds,see Molecular Probes Catalogue (1998), U.S. Pat. No. 5,631,169, issuedMay 20, 1997, U.S. Pat. No. 5,145,774, issued Sep. 8, 1992, and worldwide web site http://optics.jct.ac.il/˜aryeh/Confocal/fluoreochromes(Jul. 6, 1998) Many cnidarians use green fluorescent proteins (“GFPs”)as energy-transfer acceptors in bioluminescence. A “green fluorescentprotein,” as used herein, is a protein that fluoresces green light.Similarly, “blue fluorescent proteins” fluoresce blue light and “redfluorescent proteins” fluoresce red light. GFPs have been isolated fromthe Pacific Northwest jellyfish, Aequorea Victoria, the sea pansy,Renilla reniformis, and Phialidium gregarium (W. W. Ward et al.,Photochem. Photoobiol, 35:803-808 (1982); Levine et al, Comp. Biochem.Physiol. 72B:77-85 (1982); and Roth, Purification and ProteaseSusceptibility of the Green-Fluorescent Protein of Aequorea AequoreaWith a Note on Halistaura, Dissertation, Rutgers, The State Universityof New Jersey, New Brunswick, N.J. (1985)). GFPs have also beenengineered to be blue fluorescent proteins and yellow fluorescentproteins (U.S. Pat. No. 5,625,048 to Tsien et al., issued Apr. 29, 1997;WO 97/28261 to Tsien et al., filed Jul. 16, 1997; PCT/US 97/14593 toTsien et al., filed Aug. 15, 1997; WO 97/28261 to Tsien, published Aug.7, 1997; and WO 96/23810 to Tsien et al., published Aug. 18, 1996).

[0027] An “Aequorea-related fluorescent protein” means any protein thathas any contiguous sequence of 150 amino acids that has at least 85%sequence identity with an amino acid sequence, either contiguous ornon-contiguous, from the 238 amino acid wild-type Aequorea greenfluorescent protein of SEQ ID NO:2. More preferably, a fluorescentprotein is an Aequorea-related fluorescent protein if any contiguoussequence of 200 amino acids of the protein has at least 95% sequenceidentity with an amino acid sequence, either contiguous ornon-contiguous, from the wild type Aequorea green fluorescent protein ofSEQ ID NO:2. Similarly, the protein can be related to Renilla orPhialidium wild-type fluorescent proteins using the same standards.

[0028] A variety of Aequorea-related fluorescent proteins have beenengineered by modifying the amino acid sequence of a naturally occurringGFP from Aequorea victoria (D. C. Prasher et al, Gene, 111:229-233(1992); Heim et al. Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994);U.S. Pat. No. 5,625,048, issued Apr. 29, 1997 to Tsien et al.; WO96/23810 to Tsien, published Aug. 8, 1996; and PCT applicationPCT/US97/14593 to Tsien et al, filed Aug. 15, 1997) and have usefulexcitation and emission spectra.

[0029] A “substrate site for an activity” means a locus that is asubstrate for an activity, such as an enzymatic activity. The locus canbe any structure, such as an amino acid, chemical group or ionic orcovalent bond. Such substrate site for an activity can be part of asubstrate recognition motif that is recognized by an activity. Forexample, the site of phosphorylation within a protein is the actualamino acid modified by a phosphatase or kinase, and the site ofphosphorylation can be within a phosphorylation recognition motif. Afluorescent compound or fluorescent protein moiety can have at least onesubstrate recognition motif, which can have at least one substrate sitefor an activity. Substrate recognition motifs that are recognized byenzymatic activities are known in the art, such as for proteases,kinases, phosphatases, glycosylases, or transferases (such as famsyltransferases), or any other type of enzyme.

[0030] A “substrate recognition motif for an activity” can be anystructure or sequence that is recognized by an enzyme that directs orhelps in the enzymatic modification of the substrate by the enzyme. Thesubstrate recognition motif for an activity can be within. close to, orpart of the structure, such as amino acid residue or residues, that aremodified by the activity, such as an enzyme activity, (such as thesubstrate site for an activity). For example, the sequence surrounding aprotein kinase A phosphorylation site plays a significant role incontrolling how efficiently the site is modified. Also, protein-proteininteraction domains and protein localization domains can control theefficiency of enzymatic modifications of a substrate, such as a proteinsubstrate, and are particularly important within cells (see, Pawson etal., Science 278:2075-2080 (1997). These protein-protein interactiondomains and protein localization domains can be distal from thesubstrate recognition motif and play a role in substrate recognition.

[0031] A “fluorescent protein substrate” is a substrate for an activity,such as an enzymatic activity, that comprises a fluorescent compoundthat comprises a fluorescent protein moiety and at least one substratesite for an activity.

[0032] As used herein the term “phosphorylation recognition motif for aprotein kinase” refers to an amino acid sequence that is recognized by aprotein kinase for the attachment of a phosphate moiety. Thephosphorylation recognition motif for a protein kinase can be a siterecognized by, for example, protein kinase A, a cGMP-dependent proteinkinase, protein kinase C, Ca²⁺/calmodulin-depending protein kinase I,Ca²⁺/calmodulin-dependent protein kinase II or MAP kinase activatedprotein kinase type I, and isoforms or allelic variants thereof

[0033] As used herein, “fluorescent property” refers to the molarextinction coefficient at an appropriate excitation wavelength, thefluorescent quantum efficiency, the shape of the excitation spectrum oremission spectrum, the excitation wavelength maximum or emissionwavelength maximum, the ratio of excitation amplitudes at two differentwavelengths, the ratio of emission amplitudes at two differentwavelengths, the excited state lifetime, the fluorescent anisotropy orany other measurable property of a fluorescent compound. A measurabledifference in any one of these properties in response to a quenchingagent or under quenching conditions between a first state and a secondstate of a fluorescent compound suffices for the utility of thefluorescent compounds of the invention.

[0034] A difference in a fluorescent property of a fluorescent compoundcan be measured by determining the amount of any quantitativefluorescent property, e.g., the amount of fluorescence at a particularwavelength, or the integral of fluorescence of the emission spectrum.Determining ratios of excitation amplitude or emission amplitude at twodifferent wavelengths (“excitation amplitude ratioing” and “emissionamplitude ratioing,” respectively) are particularly advantageous becausethe ratioing process provides an internal reference an cancels outvariations in the absolute brightness of the excitation source, thesensitivity of the detector, and light scattering or quenching by thesample. Furthermore, if a change in a fluorescent compound from a firststate to a second state changes the fluorescent compound's ratio ofexcitation or emission amplitudes at two different wavelengths, thensuch ratios measure the extent of the first state and second stateindependent of the absolute quantity of the fluorescent compound.

INTRODUCTION

[0035] The present invention recognizes that fluorescent compounds, suchas fluorescent proteins, can exist in at least two states and that thefluorescent properties of these two states can be different, preferablyunder quenching conditions, and reflect different biochemical orchemical characteristics of the fluorescent compounds. Particularly, thestability of the fluorescence in one state can be different from thestability of the fluorescence in the second state, which can be detectedby a sensitivity to quenching The conversion of a first state to asecond state of the fluorescent compound can be caused by the action ofa chemical or enzyme, such as a protease, kinase, phosphatase,glycosylase, transferase such as protein prenyl transferase, or anyother enzyme that can modify the fluorescent compound. Binding ofmoieties or hybridization (in the case of nucleic acids such as DNA orRNA) can also alter the fluorescence properties of a fluorescentcompound after quenching. Thus, the present invention recognizes thatsuch fluorescent compounds can be used to detect various enzymaticactivities in a sample, such as in a cell, a cell culture, a cellextract, conditioned medium, or in an array. Such hybridization orbinding can occur and be detected in high-density arrays or on genechips such as they are known in the art (See, Johnson. Curr. Biol.8:R171-4 (1998); Livache et al., Anal. Biochem. 255:188-194 (1998)). Thepresent invention recognizes that fluorescent compounds can befluorescent proteins and that these fluorescent proteins can beexpressed in cells. Thus, the fluorescent proteins can be used as invivo monitors of intracellular enzymatic activity and used to screencompounds for drugs that modulate an enzymatic activity. The presentinvention also provides nucleic acid molecules that encode fluorescentproteins that exhibit quenching.

[0036] As a non-limiting introduction to the breath of the invention,the invention includes several general and useful aspects, including:

[0037] 1) A fluorescent compound for detecting an activity, comprising afluorescent protein and a substrate recognition motif for an activity.wherein said fluorescent compound exhibits quenching,

[0038] 2) A nucleic acid molecule coding for the expression of thefluorescent compound in 1),

[0039] 3) A cell comprising the nucleic acid molecule of 2) orfluorescent compound of I),

[0040] 4) A method for determining whether a sample contains anactivity, comprising contacting a sample with a fluorescent compound of1), exciting said fluorescent compound, and measuring the amount ofemission from said fluorescent compound under quenching conditions, and

[0041] 5) A method for determining whether a cell exhibits an activitycomprising exciting a transfected host cell comprising a recombinantnucleic acid molecule of 2) or fluorescent compound of 1). and measuringthe emission from the fluorescent compound under quenching conditions.

[0042] Fluorescent Compounds for Detecting an Activity.

[0043] The present invention provides fluorescent compounds fordetecting an activity, comprising: a fluorescent protein and at leastone substrate site for an activity, wherein said fluorescent compoundcan exist in at lease two states that exhibit quenching.

[0044] The sensitivity of the fluorescent compound to quenching isinfluenced by a modification of the fluorescent compound from a firststate to a second state by an activity, such as an enzymatic activity.Such modification of the fluorescent compound can stabilize ordestabilize the fluorescent compound by, for example, changing theconformation of the fluorescent compound, the addition of a moiety tothe fluorescent compound, or the removal of a moiety from thefluorescent compound. Exposure of the fluorescent compound to chemicalsor enzymes can cause such modifications.

[0045] For example, a ligand binding to a fluorescent compound can altera fluorescent property of the fluorescent compound, making thefluorescent compound more or less sensitive to quenching. Likewise, theassociation of moieties with a fluorescent compound (by, for example,chemical or enzymatic reaction) or the aggregation of fluorescentcompounds., or hybridization of nucleic acids, can alter at least onefluorescent property of a fluorescent compound that can be detected byquenching. Furthermore, moieties can be added to a fluorescent compoundby covalent modification that can result in an altered fluorescentproperty of the fluorescent compound that can be detected by quenching.Likewise, covalent bonds can be cleaved within the structure of thefluorescent compound that can result in an altered fluorescent propertyof the fluorescent compound that can be detected after quenching.

[0046] Covalent bonds can be made or broken by well-known chemical orenzymatic reactions. For example, peptide bonds can be hydrolyzed byacidic conditions or by proteases. Also, phosphate groups can be addedto a fluorescent compound by kinases, and removed by phosphatases. Otherenzymatic reactions, such as lipases, the addition or loss of lipidmoieties such as farnesyl, geranylgeranyl or phosphatidyl inositolgroups, and the like can be used to modify fluorescent compounds, whichcan in turn alter a fluorescent property of the fluorescent compoundthat can be measured by quenching.

[0047] A change in the number or distribution of disulfide bonds by analteration in the redox state thereof can also alter a fluorescentproperty of a fluorescent compound. For example, reducing agents, suchas beta-mercaptoethanol, can alter the oxidation state of disulfidebonds within a fluorescent compound having such structures. The changein the structure of the fluorescent compound caused by such treatmentcan cause a change in a fluorescent property of the fluorescent compoundthat can be detected after quenching.

[0048] When an activity can modify a fluorescent compound, thefluorescent compound can comprise a naturally occurring substraterecognition motif (for example, endogenous to the fluorescent compound)for such enzymatic reactions, or such substrate recognition motifs canbe added or engineered into the fluorescent compound (for exampleexogenous to the fluorescent compound). For example, a fluorescentcompound that is a protein can be engineered to comprise a substraterecognition motif for a protease, protein phosphatase, protein kinase,protein prenyltransferase, glycosylase, or any other enzyme usingmethods known in the art. For example, genetic engineering, chemicalmodification techniques, or enzyme reactions can be used to add suchsubstrate recognition motifs to the amino- or carboxy-terminus of afluorescent compound. Alternatively, these techniques can be used toinsert such substrate recognition motifs within the structure of thefluorescent compound.

[0049] A change in the tertiary structure of a fluorescent compound bythe addition or removal of a moiety by chemical or enzymatic activitycan also cause a change in a fluorescent property of a fluorescentcompound after quenching. For example, phosphorylation of a fluorescentcompound can lead to a change in its tertiary structure through thecreation of new or stronger interactions between amino acid residues,which can result in stabilized fluorescence that can result in increasedresistance to quenching. Such a change in sensitivity to quenching cansubsequently be used to measure the amount of fluorescent compound thathas been phosporylated, and hence the activity of the kinase can bedetected and/or measured. Such moieties can also destabilize thetertiary structure of a fluorescent protein, which can result indestabilized fluorescence under quenching conditions. Furthermore,enzymatic activities such as proteases can alter the tertiary structureof a fluorescent protein, which can also result in destabilizedfluorescence under quenching conditions. Furthermore, the presence of anelectrochemical, chemical or electrical gradient or potential can alsochange a fluorescent property of a fluorescent compound that can bedetected through quenching.

[0050] Quenching agents can be used to detect the stabilization,destabilization, or protection of a fluorescent property of afluorescent compound arising from an activity. For example, fluorescentcompounds such as fluorescent proteins can be stabilized or destabilizedby acidic conditions, basic conditions, surfactants, organic solvents,chaotropic salts or agents, anti-chaotropic salts or agents, reducingconditions or agents, oxidizing conditions or agents, paramagnetic ions,enzymes, collisional quenchers? temperature or any combination thereof.

[0051] Fluorescent compounds, such as fluorescent proteins, can be madeusing methods known in the art. For example, fluorescent proteins can bemade by expressing nucleic acids that encode fluorescent proteins, suchas wild-type or mutant Aequorea green fluorescent protein, or otherfluorescent proteins such as those from Renilla, in an appropriatecellular host (WO 96/2381 to Tsien et al., published Aug. 18, 1996).Alternatively, proteins that are otherwise not fluorescent can be madefluorescent by covalently linking or binding a fluorophore, such asfluorescein, to the protein using methods known in the art (U.S. Pat.No. 5,605,809 to Kumoriya et al., issued Feb. 25, 1997).

[0052] Fluorescent compounds, such as fluorescent proteins, can havestructures that allow them to be altered from a first state to a secondstate by, for example, binding of a ligand, post translationalmodifications such as phosphorylation, dephosphorylation, proteolysis,or glycosylation at specific sites. Compounds can be modified to includesuch specific sites using methods known in the art. For example,fluorescent proteins, such as Aequorea green fluorescent protein, can beengineered to have non-naturally occurring substrate recognition motifs,such as known phosphorylation or protease motifs and sites. Thephosphorylation or proteolytic status of the fluorescent compound canalter the stability of the compound such that the phosphorylated,unphosphorylated, or proteolytic states of the fluorescent compound canbe detected or measured using quenching.

[0053] Optimal alignment of sequences for aligning a comparison windowmay be conducted by the local homology algorithm of Smith and Waterman(Adv. Appl. Math, 2:482 (1981)) by the homology alignment algorithm ofNeedleman and Wunsch (J. Mol. Biol. 48-:443 (1970)) by the search forsimilarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA85:2444 (1988)) by computerized implementations of algorithms GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package(Release 7.0. Genetics Computer Group, Madison, Wis.) or by inspection.The best alignment, (i.e. resulting in the highest percentage ofhomology over the comparison window, i.e., 150 or 200 amino acids)generated by the various methods is preferably selected. The percentageof sequence identity is calculated by comparing two optimally alignedsequences over the window of comparison, determining the number ofpositions at which the identical amino acid occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison(i.e. the window size), and multiplying the results by 100 to yield thepercentage of sequence identity.

[0054] Aequorea-related fluorescent proteins include, for example, andwithout limitation, wild-type (native) Aequorea victoria GFP (Prasher,Gene 11 1:229-233 (1992). whose nucleotide sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2), allelic variants or othervariants of this sequence (for example, Q80R, which has the glutimineresidue at position 80 substituted with arginine (Chalfie et al,Science, 263:802-805 (1994)), those Aequorea-related engineered versionsdescribed in TABLE I, variants that include one or more folding mutantsand fragments of these proteins that are fluorescent, such as Aequoreagreen fluorescent protein form which the two amino-terminal amino acidshave been removed from the amino- or carboxy-terminus. Several of thesecontain different aromatic amino acids within the central chromophoreand fluoresce at a distinctly shorter wavelength than wild type species.For example, mutants P4 and P4-3 contain, in addition to othermutations, the substitution Y66H. Mutants W2 and W7 contain, in additionto other mutations, Y66W. Other mutations, provided as non-limitingexamples are listed in TABLE I. The following six groups each containamino acids that are conserved substitutions for one another.

[0055] 1) Alanine (A), Serine (S), Threonine (T),

[0056] 2) Aspartic Acid (D), Glutamic Acid (E),

[0057] 3) Asparagine (N), Glutamine (O),

[0058] 4) Arginine (R), Lysine

[0059] 5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V), and

[0060] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0061] Other mutations are set forth in U.S. Pat. No. 5,625,048 to Tsienet al. issued Apr. 29, 1997; WO 96/23810 to Tsien et al., published Aug.18, 1996; PCT/US 97/12410 to Tsien et al., filed Jul. 16, 1997; PCT/US97/14593 to Tsien et al., filed Aug. 15, 1997; and PCT/US 96/04059 toTsien et al., filed Mar. 20, 1996. TABLE I Fluorescent mutants ofAequorea green fluorescent protein Excita- Emis- Extinction tion sionCo- Quan- Muta- Max Max efficient tum Clone tion(s) (nm) (nm) (M-1cm-1)Yield Wild None 395 508 21,000 0.77 type (475) (7,150) P4 Y66H 383 44713,500 0.21 P4-3 Y66H 381 445 14,000 0.38 Y145F W7 Y66W 433 475 18,0000.67 N1461 (453) (501) (17,100) M153T V163A N212K W2 Y66W 432 480 10,0000.72 I123V (453) (9,600) Y145H H148R M153T V163A N212K S65T S65T 489 51139,200 0.68 P4-I S65T 504 514 14,500 0.53 M153A (396) (8,600) K238E S65AS65A 471 504 S65C S65C 497 507 S6SL S65L 484 510 Y66F Y66F 360 442 S65TS65T 489 511 39,200 0.68 Y66H Y66H 382 448 Y66W Y66W 458 480 K4 SEQ ID471 505 NO:.49 −2M −1G M1R S2R K3R G4A E5S E61 L71 S65A N149K V163AI1671 K5 K4 + K79N 471 505 K6 K4 + E90N 471 505 K7 K4 + E90K 471 505 K8K4 + K79R 471 505 E90N K9 K4 + K79R 471 505 E90K K10 K4 + K79H 471 505E90N K11 K4 + K79H 471 505 E90K K12 K4 + K79H 471 505 K13 K4 + K79E 471505 E90N K14 K4 + K79E 471 505 E90K K15 K4 + K79E 471 505 K16 K4 + K79Q471 505 K4 + E90A K4 + E90A 471 505 K4 + E90L K4 + E90L 471 505 K4 +E90V K4 + E90V 471 505 K4 + E90T K4 + E90T 471 505 K4 + E90S K4 + E90S471 505 K4 + E901 K4 + E901 471 505 K4 + K79R K4 + K79R 471 505 1167T1167T 471 502 T2031 T2031 H9 S202F 398 511 T2031 P9 I167V 471 502 (396)(507) P11 I167T 471 502 (396) (507) 10C S65G 513 527 V68L S72A T203Y W1BF64L 432 476 S65T (453) (503) Y66W N1461 M153T V163A N212K Emerald S65T487 508 S72A N149K M153T I167T Topaz S65G 514 528 S72A K79R T203Y P4-SEY66H 382 446 Y145F F64L V163A Sapphire S72A 395 571 Y145F T2031

[0062] Additional mutations in Aequorea-related fluorescent protein,referred to as “folding mutations,” improve the ability of GFP to foldat higher temperatures, and to be more fluorescent when expressed inmammalian cells, but can have little effect on the peak wavelengths ofexcitation and emission. It should be noted that these folding mutantscan be combined with mutations that influence the spectral properties ofGFP to produce proteins with altered spectral and folding properties.Folding mutations include the following mutations: T44A, F64L,V68L,S72A, F99S, Y145F, N1461, S147P, M153T or A, V163A, 1167T, S175G,S205T and N212K.

[0063] This invention contemplates the use of other fluorescent proteinsin fluorescent protein substrates. The cloning and expression of yellowfluorescent protein from Vibrio fisheri strain YU-1 has been describedby Baldwin et al. (Biochemistry 29:5509-5515 (1990)). This proteinrequires flavins as fluorescent cofactors. The cloning of Peridininchlorophill a binding protein from the dinoflagellate Symbioclinium sp.,was described by Morris et al, (Plant Molecular Biology 24:673-677(1994)). One useful aspect of this protein is that is fluoresces red.The cloning of the phycobiliprotiens from marine cyanobacteria such asSynechoccus, e.g., phycoerythrin and phycocyanin, is described inWilbands et al., (J. Biol. Chem. 268:1226-12235 (1993). These proteinssequence phycobilins as fluorescent co-factors, whose insertion into theproteins involves auxiliary enzymes. These protein fluoresce at yellowto red wavelengths.

[0064] It has been found that fluorescent proteins can be geneticallyfused to other proteins and used as markers to identify the location andamount of the target protein produced. Accordingly, this inventionprovides fusion proteins comprising a fluorescent protein moiety andadditional amino acid sequences such as amino acid sequences encoding aprotein or polypeptide or peptide of interest. Such additional aminoacid sequences can be, for example, up to about 15, up to about 150 orup to about 1,000 amino acids long and comprise a substrate site for anactivity, such as an enzymatic activity. The fusion proteins possess theability to fluorescence when excited by electromagnetic radiation. Inone embodiment, the fusion protein comprises a polyhistidine tag to aidin purification of the protein or a poly-Lys portion to aid in membraneassociation of the fluorescent compound.

[0065] Fluorescent protein substrates for a protein kinase are thesubset of fluorescent proteins as defined above whose amino acidsequence includes a phosphorylation recognition motif and site.Fluorescent protein substrates can be made by modifying the amino acidsequence of an existing fluorescent protein to include a phosphorylationrecognition motif and site for a protein kinase.

[0066] A consensus phosphorylation recognition motif for protein kinaseA is RRXSZ (SEQ ID NO:3) or RRXTZ (SEQ ID NO:4), wherein X is any aminoacid and Z is a hydrophobic amino acid, preferably valine, leucine orisoleucine. Many variations in the above sequence are allowed, butgenerally exhibit poorer kinetics. For example lysine (K) can besubstituted for the second arginine. Many consensus sequences for otherprotein kinases have been tabulated (e.g. by Kemp and Pearson, TrendsBiochem. Sci. 15:342-346 (1990); Songyang et al., Current Biology4:973-982 (1994); Nishikawa et al., J. Biol. Chem. 272952-960 (1997);and Songyang et al., Mol. Cell. Biol. 16:6486-6493 (1996)).

[0067] For example, a fluorescent protein substrate selective forphosphorylation by cGMP-dependent protein kinase can include thefollowing consensus phosphorylation recognition motif sequence:BKISASEFDRPLR (SEQ ID NO:5), where B represents either lysine (K) orarginine (R), and the first S is the site of phosphorylation (Colbran etal, J. Biol. Chem. 267:9589-9594 (1992)). The residues DRPLR (SEQ IDNO:6) are less important than the phenylalanine (F) just preceding themfor specific recognition by cGMP-dependent protein kinase in preferenceto cAMP-dependent protein kinase.

[0068] Either synthetic or naturally occurring phosphorylationrecognition motifs can be used to create a protein kinasephosphorylation site. For example, peptides including the motif XRXXSXRX(SEQ ID NO:7), wherein X is any amino acid, are among the best syntheticsubstrates (Kemp and Pearson, supra) for protein kinase C.Alternatively, the Myristoylated Alanine-Rich Kinase C substrate(“MARCKS”) is one of the best substrates for PKC and is an efficientreal target for the kinase in vivo. The Examples set forth additionalsubstrates for PKC. The phosphorylation recognition motif sequencearound the phosphorylation site of MARCKS is KKKKRFSFK (SEQ ID NO:8)(Graffet al., J. Biol. Chem. 266:14390-14398 (1991)). Either of thesetwo sequences can be incorporated into a fluorescent protein to make ita substrate for protein kinase C.

[0069] A protein substrate for Ca²⁺/calmodulin-dependent protein kinaseI is derived from the sequence of synapsin, a known optimal substratefor this kinase. The phosphorylation recognition motif around thephosphorylation site is LRRLSDSNF (SEQ ID NO:9) (Lee et al., Proc. Natl.Acad. Sci. USA 91:6413-6417 (1994).

[0070] A protein substrate selective for Ca²⁺/calmodulin-dependentprotein kinase II is derived from the sequence of glycogen synthase, aknown optimal substrate for this kinase. The recognition sequence aroundthe phosphorylation site is KKLNRTLTVA (SEQ ID NO:10) (Stokoe et al.Biochem. J. 296:843-849 (1993)). A small change in this sequence toKKANRTLSVA (SEQ ID NO: 11) makes the latter specific for MAP kinaseactivated protein kinase type 1. Other preferred kinase recognitionmotifs and sites are provided in TABLE II below. One skilled in the artwould realize that many proteins that do not contain such preferredphosphorylation motifs and sites can be phosphorylated if they conformto the consensus motif, but that the rates of phosphorylation can beless than for the preferred substrates. TABLE II Underlined residue isphosphorylation site. SEQ ID NO Protein Kinase Sequence SEQ ID NO. 50Cyclin B-CDC2 HHHKSPRRR SEQ ID NO. 51 Cyclin A-CDK2 HHHRSRPKR SEQ ID NO.52 Protein Kinase A RRRRSIIFI SEQ ID NO. 53 SLK I RRFGSLRRL SEQ ID NO.54 ERK I TGPLSPGPF SEQ ID NO. 55 Protein Kinase Cα RRRRRKGSFRRKA

[0071] In one embodiment, the fluorescent protein substrate contains aphophorylation motif and site at or about one or more of the termini, inparticular, the amino-terminus, of the fluorescent protein moiety. Thesite preferably is located in a position within five, ten, fifteen, ortwenty amino acids of a position corresponding to the wild typeamino-terminal amino acid of the fluorescent protein moiety. Thisincludes sites engineered into the existing amino acid sequence of thefluorescent protein moiety and can also be produced by extending theamino terminus of the fluorescent protein moiety.

[0072] One may, for example, modify the existing sequence of wild typeAequorea GFP, or a variant thereof, to include a phosphorylation sitewithin the first ten, twelve, fifteen, eighteen or twenty amino acids ofthe N-terminal met of wild-type Aeqiorea GFP (or its equivalent in afusion protein). In one embodiment, the naturally occurring sequence ismodified as follows: Wild type: MSKGEELFTG residues (1 to 10 of SEQ IDNO:2) Substrate: MRRRRSIITG. (SEQ ID NO:12)

[0073] One may include modifying the naturally occurring sequence ofAequorea GFP by introducing a phosphorylation motif or site into anextended amino acid sequence of such a protein created by addingflanking sequences to the amino terminus, for example: Wild type:MSKGEELFTG residues (1 to 10 of SEQ ID NO:2) Substrate MRRRRSIIIIFTG.(SEQ ID NO:13)

[0074] Fluorescent protein substrates having a phosphorylation site ator about a terminus of a fluorescent protein moiety offer the followingadvantages. First, it is often desirable to append additional amino acidresidues onto the fluorescent protein moiety to create a specificphosphorylation consensus sequence. Such a sequence is less likely todisrupt the folding pattern of a fluorescent protein moiety whenappended onto the terminus than when inserted into the interior of theprotein sequence. Second, different phosphorylation motits can beinterchanged without significant disruption of GFP, thereby providing ageneral method of measuring different kinases. Third, thephosphorylation site is preferably exposed to the surface of the proteinand, therefore, more accessible to protein kinases. Fourth, we havediscovered that phosphorylation at sites close to the N-terminus of GFPcan provide large changes in fluorescent properties if the site ofphosphorylation is chosen such that the Ser or Thr residue that isphosphorylated occupies a position that was originally negative orpositively charged in the wild-type protein. Specifically. replacementof Glu 5 or Glu 6 by a non-charged Ser or Thr residue can significantlydisrupt the fluorescence, folding, or sensitivity of GFP to quenching.Phosphorylation of the serine or threonine can restore negative chargeto this position and thereby increases the ability of GFP to foldcorrectly at higher temperatures, and hence can increase thefluorescence of GFP and resistance to quenching.

[0075] In another embodiment, the fluorescent protein substrate includesa phosphorylation site remote form a terminus, e.g., that is separatedby more than about twenty amino acids from the terminus of theflorescent protein moiety and within the fluorescent protein moiety. Oneembodiment of this form includes the Aequorea-related fluorescentprotein substrate comprising the substitution H217S, creating aconsensus protein kinase A phosphorylation motif and site. Additionally,phosphorylation recognition motifs comprising the following alterationsbased on the sequence of wild type Aequorea GFP exhibit fluorescentchanges upon phosphorylation: 69RRFSA (SEQ ID NO: 14) and 214KRDSM (SEQID NO:15) (which includes H217S).

[0076] The artisan should consider the following when selecting aminoacids for substitution within the fluorescent protein moiety remote inprimary amino acid sequence form the terminus. First, it is preferableto select amino acid sequences within the fluorescent protein moietythat resemble the sequence of the phosphorylation motif and site. Inthis way, fewer amino acid substitutions in the native protein areneeded to introduce the phosphorylation motif and site into thefluorescent protein. For example, protein kinase A recognizes thesequence RRXSZ (SEQ ID NO:3) or RRXTZ (SEQ ID NO:4), wherein X is anyamino acid and Z is a hydrophobic amino acid. Ser or Thr is the site ofphosphorylation. It is preferable to introduce this sequence into thefluorescent protein moiety at sequences already containing Ser or Thr sothat Ser or Thr are not substituted in the protein. More preferably, thephosphorylation recognition motif is created at locations having someexisting homology to the sequence recognized by protein kinase A, e.g.having a proximal Arg or hydrophobic residues with the same spatialrelationship as in the phosphorylation site.

[0077] Second, location on the surface of the fluorescent protein ispreferred for phosphorylation sites. This is because surface locationsare more likely to be accessible to protein kinaes than interiorlocations. Surface locations can be identified by computer modeling ofthe fluorescent protein structure or by reference to the crystalstructure of Aequorea GFP. Also, charged amino acids or groups ofcharged amino acids in the fluorescent protein are more likely to lie onthe surface than inside the fluorescent protein, because such aminoacids are more likely to be exposed to water in the environment.

[0078] In cases where the phosphorylation site is either at a terminus,such as the N-terminus, or remote from a terminus, the amino acidcontext around the phosphorylation site can be optimized in order tomaximize the change in fluorescence. Amino acid substitutions thatchange large bulky and/or hydrophobic amino acids to smaller and lesshydrophobic replacements are generally helpful. Similarly, large chargedamino acids can be replaced by smaller, less charged amino acids. Forexample:

[0079] a) Hydrophobic to less hydrophobic

[0080] Phe to Leu

[0081] Leu to Ala

[0082] b) Charged to charged but smaller

[0083] Glu to Asp

[0084] Arg to Lys

[0085] c) Charged to less charged

[0086] Glu to Gln

[0087] Asp to Asn

[0088] d) Charged to polar

[0089] Glu to Thr

[0090] Asp to Ser

[0091] e) Changed to non-polar

[0092] Glu to Leu

[0093] Asp to Ala

[0094] These changes can be accomplished by direct means or using randomiterative approaches where changes are made randomly and the best onesselected based upon their change in fluorescent properties afterphosphorylation by an appropriate kinase.

[0095] Third, amino acids at distant locations from the actual site ofphosphorylation can be varied to enhance fluorescence changes uponphosphorylation. These mutations can be created through site directedmutagenesis, or through random mutagenesis, for example by error-pronePCR, to identify mutations that enhance either absolute fluorescence orthe change in fluorescence upon phosphorylation. The identification ofmutants remote in primary sequence from a terminus, such as anN-terminus, identifies potentially interacting sequences that mayprovide additional areas in which further mutagenesis can be used torefine the change in fluorescence upon phosphorylation. For example,mutations around the amino terminus phosphorylation site may interact(either transiently during folding, or in a stable fashion) with aminoacids at positions 171 and 172, and point mutations that significantlydisrupt fluorescence of GFP by changing negative to positive chargesnear the amino terminus can be rescued by changing a positive to anegative charge at position 171.

[0096] In the phosphorylation mutant below the sequence is a), the wildtype sequence b) is also listed below. a) MSKRRDSLT (SEQ ID NO:16) b)MSKGEELFT (1 to 9 of SEQ ID NO:2)

[0097] The phosphorylation mutant has only 7% of the fluorescence of thewild type protein. However, its fluorescence can be restored to 80% ofthe wild type by two amino acid changes, E171K and I172V, positionswhich are quite remote in linear sequence form the amino terminus.

[0098] Thus, changes in charge at E171 K (negative to positive) canalmost completely restore the fluorescence of the phosphorylationmutant, strongly suggesting that the original loss of fluorescence aroseprimarily through changes in charge caused by the point mutations. It isclear that the addition and loss of charge at positions around, and atthe phosphorylation site, have a significant impact on fluorescenceformation. The fact that charge alone can significantly affect thefluorescence properties of GFP is highly significant within the scope ofthe present application since phosphorylation involves the addition oftwo negative changes associated with the phosphate group on the serineresidue.

[0099] In the above case the mutations restore fluorescence of thephosphorylation mutant without significantly increasing the magnitude ofthe change in fluorescence upon phosphorylation. Nevertheless, theidentification of these positions in GFP provides a valuable tool tofurther enhance changes in fluorescence upon phosphorylation by creatingrandom mutations at codons around positions 171, 172, and 173 toidentify mutations that enhance changes in fluorescence uponphosphorylation. This can be achieved by co-expressing the kinase ofinterest with the fluorescent substrate of the present inventioncontaining random mutations that may enhance the fluorescence changesupon phosphorylation.

[0100] A GFP-based phosphorylation sensor having a phosphorylation motifor site at or near the amino-terminus can be modified to establish aphosphorylation sensor having enhanced fluorescence, enhanced kineticsof phosphorylation, and enhanced changes in fluorescence upon quenching.Within the amino terminal sequence of GFP the first four amino acids arefreely interchangeable. The next seven amino acids can be modified,preferably with conservative amino acid changes, to accommodate aphosphorylation recognition motif. To achieve high levels offluorescence, it may be preferable to mutate addition sites in GFP topromote efficient folding. These methods are discussed in the Examples.In addition to enhancing fluorescence of such sensors, the kinetics ofphosphorylation of these sensors can be enhanced. For example, theefficiency with which a phosphorylation site is modified by a kinase orphosphatase is dependent on the sequence and accessibility of therecognition motif. The accessibility of the phospohrylation motif can beimproved my making changes in amino acids that disorder the localamino-terminal structure of GFP or reduce interactions between theamino-terminal region and the interior of the molecule, preferably bydisrupting interactions between Lys3 and Glu90 and amino acids aroundthese residues.

[0101] Preferable mutants can be identified using, for example, thefollowing method. Nucleic acid molecules encoding such fluorescentcompounds, such as kinase sensors, can be placed into an appropriateexpression vector. The expression vector can also encode an activity,such as an activity specific for the fluorescent compound, such as akinase. The expression vectors are transformed into host cells, such asbacteria or mammalian cells, such as human cells, and the individualbacterial colonies grown up. to Each colony is derived from a singlecell, and hence contains a single unique mutant fluorescent substrategrown up. The individual colonies may then be grown up and screened forfluorescence either by fluorescence activated cell sorting (FACS), or byobservation under a microscope. Those that exhibit the greatestfluorescence can then be screened under conditions in which the geneencoding the activity, such as a kinase activity, is inactivated.Appropriate digests of the kinase gene can achieve this by restrictionenzymes that specifically cut within the kinase but not GFP. Comparisonof the brightness of the mutant first in the presence of kinase then inits absence indicates the relative effect of phosphorylation of themutant GFP.

[0102] Fluorescent protein substrates for a protease can be made usingthe methods and strategies discussed above for fluorescent proteinsubstrates for a protein kinase. The skilled artisan need merelyincorporate a protease cleavage recognition site into the fluorescentcompound rather than a substrate site for a protein kinase. Suchprotease cleavage recognition sites are known in the art, some of whichare presented in TABLE III. TABLE III Protease Sequence HIV-1 proteaseSQNY-PIVQ (SEQ ID NO: 37) KARVL-AEMS (SEQ ID NO: 38) Prohormoneconvertase PSPREGKR-SY (SEQ ID NO: 39) Interleukin-1b-converting enzymeYVAD-G (SEQ ID NO.: 40) Adenovirus endopeptidase MFGG-AKKR (SEQ ID NO:41) Cytomegalovirus assemblin GVVMA-SSRLA (SEQ ID NO: 42) LeishmanolysinLIAYI-LKKAT (SEQ ID NO: 43) b-Secretase for amyloid precursor proteinVKM-DAEF (SEQ ID NO: 44) Thrombin FLAEGGGVR-GPRVVERH (SEQ ID NO: 45)DRVYIHPF-HL-VIH (SEQ ID NO. 46) Renin and angiotensin-converting EnzymeCathepsin D KPALF-FRL (SEQ ID NO: 47) Kininogenases including kallikreinQPLGQTSLMK-RPPGFSPER SVQVMKT QEGS (SEQ ID NO: 48)

[0103] See, e.g., Matayoshi et al. (1990) Science 247:954, Dunn et al.(1994) Meth. Enzymol. 241:254, Seidah I & Chretien (1994) Meth. Enzymol.244:175, Thomberry (1994) Meth. Enzymol. 244:615, Weber & Tihanyi (1994)Meth. Enzymol. 244:595, Smith et al.(1994) Meth. Enzymol. 244:412,Bouvier et al. (1995) Meth. Enzymol. 248:614, Hardyi et al. (1994) inAmyloid Protein Precursor in Development, Aging, and Alzheimer'sDisease,

[0104] The methods discussed above can be used to confirm that afluorescent compound comprising a protease cleavage motif and siteexhibits at least one different fluorescent property in the cleaved anduncleaved state In addition to protein kinase substrates, proteinphosphatase substrate, and protease substrates, the present inventionencompasses substrates for protein prenyltransferases, glycosylases, anyother enzyme recited in this application, any other known enzyme, or anyenzyme later discovered. Fluorescent compounds that are substrates forthese enzyme activities can be made using the methods described in thepresent application following the exemplary teachings set forth in theExamples. For example, different substrate recognition motifs andsubstrate sites for activities, such as enzyme activities, can beincorporated into GFP to make and confirm that compounds that canexhibit quenching in response to an activity. Furthermore, fluorescentcompounds other than GFP that have at least one substrate recognitionmotif and site for activities can be made and confirmed to exhibitquenching in response to an activity by following the exemplaryteachings set forth in the Examples.

[0105] Furthermore, the present invention encompasses substrate sitesfor an activity, such as an enzymatic activity, that exhibits at leastone different fluorescent property in a first and second state afterquenching that can be detected by fluorescent detection methods. Suchactivities include proteases, transferases, glycosylases, reductases,oxidases, or any other enzyme recited in this application, any otherknown enzyme, or any enzyme later discovered. Such substrates can bemade using the exemplary teachings set forth in the Examples.

[0106] Nucleic Acid Molecules Coding for the Expression of a FluorescentCompound

[0107] While certain florescent compounds can be prepared chemically,for example, by coupling a fluorescent moiety to the amino terminus of aprotein moiety, it is preferable to produce fluorescent compoundscomprising a peptide or protein recombinantly.

[0108] Recombinant production of a fluorescent compound involvesexpressing a nucleic acid molecule having sequences that encode apeptide or protein. As used herein, the term “nucleic acid molecule”includes both DNA and RNA molecules. It will be understood that when anucleic acid molecule is said to have a DNA sequence, this also includesRNA molecules having the corresponding RNA sequence in which “U”replaces “T.” The term “recombinant nucleic acid molecule” refers to anucleic acid molecule which is not naturally occurring, and whichcomprises two nucleotide sequences that are not naturally joinedtogether. Recombinant nucleic acid molecules are produced by artificialcombination, e.g., genetic engineering techniques or chemical synthesis.

[0109] In one embodiment, the nucleic acid encodes a fusion protein inwhich a single polypeptide includes the fluorescent protein moietywithin a longer polypeptide In another embodiment, the nucleic acidencodes the amino acid sequence that comprises a substrate site for anactivity consisting essentially of a fluorescent protein moiety modifiedto include a substrate site for an activity. In either case, nucleicacids that encode fluorescent proteins are useful as starting materials.

[0110] Nucleic acids encoding a fluorescent protein moiety can beobtained by methods known in the art. For example, a nucleic acidencoding a GFP can be isolated by polymerase chain reaction of cDNA fromA. victoria using primers based on the DNA sequence of A. victoria greenfluorescent protein (SEQ ID NO:2). PCR methods are described in, forexample, U.S. Pat. No. 4,683,195; Mullis et al, cold Spring Harbor Symp.Quant. Biol. 51:263 (1987). and Erlich, PCR Technology, Stockton Press,NY (1989).

[0111] Mutant versions of fluorescent proteins can be made bysite-specific mutagenesis of other nucleic acids encoding a fluorescentprotein moiety or by random mutagenesis caused by increasing the errorrate of PCR of the original polynucleotide with 0.1 mM MnCl₂ andunbalanced nucleotide concentrations (U.S. Pat. No. 5,625,048 to Tsien,issued Apr. 29, 1997; and PCT/US95/14692, filed Nov. 10, 1995).

[0112] Nucleic acids encoding fluorescent compounds that are fusionsbetween a polypeptide including a phosphorylation site and a fluorescentprotein moiety can be made by ligating nucleic acids that encode each ofthese. Nucleic acids encoding fluorescent compounds that include theamino acid sequence of a fluorescent protein moiety in which one or moreamino acids in the amino acid sequence of a fluorescent protein moietyare substituted to create a substrate site for an activity can becreated by, for example, site specific mutagenesis of a nucleic acidencoding a fluorescent protein moiety.

[0113] Nucleic acids used to transfect cells with sequences coding forexpression of a polypeptide of interest such as those encoding afluorescent compound generally will be in the form of an expressionvector including expression control sequences operatively linked to anucleotide sequence coding for expression of the polypeptide. As usedherein, the term “nucleotide sequence coding for expression of apolypeptide” refers to a sequence that, upon transcription andtranslation of mRNA, produces the polypeptide. As any person skilled inthe art recognizes, this includes all degenerate nucleic acid sequencesencoding the same amino acid sequence. This can include sequencescontaining, e.g. introns. As used herein, the term “expression controlsequences” refers to nucleic acid sequences that regulate the expressionof a nucleic acid sequence to which it is operatively linked. Expressioncontrol sequences are “operatively linked” to a nucleic acid sequencewhen the expression control sequence control and regulate thetranscription and, as appropriate, translation of the nucleic acidsequence. Thus, expression control sequences can include appropriatepromoters, enhancers, transcription termination, or a start codon.(i.e., ATG) in front of a protein-encoding gene, splicing signals forintrons, maintenance of the correct reading frame of that gene to permitproper translation of the mRNA, and stop codons. Recombinant nucleicacid can be incorporated into an expression vector comprising expressioncontrol sequences operatively linked to the recombinant eukaryotes byinclusion of appropriate promoters, replication sequences. market, etc.The expression vector can be transfected into a host cell for expressionof the recombinant nucleic acid. Host cells can be selected for highlevels of expression in order to purify the protein. E. coli is usefulfor this purpose. Alternatively, the host cell can be a prokaryotic oreukaryotic cell selected to study the activity of an enzyme produced bythe cell. The cell can be, e.g. a cultured cell or a cell in vivo. Theconstruction of expression vectors and the expression of genes intransfected cells involves the use of molecular cloning techniques alsowell known in the art (Sambrook et al., Molecular Cooing—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989);Ausubel et la., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, Inc.).

[0114] Recombinant fluorescent protein substrates can be produced byexpression of nucleic acid encoding for the protein in E. coli.Aequorea-related florescent proteins are best expressed by cellscultured between about 15° C. and 30° C. but higher temperatures (e.g.37° C.) are possible. After synthesis, these enzymes are stable athigher temperatures (e.g. 37° C.) and can be used in assay at thosetemperatures.

[0115] The construct can also contain a tag to simplify isolation of theexpressed fluorescent compound. For example, a polyistidine tag of, e.g.six histidine residues, can be incorporated at the amino or carboxylterminal of the fluorescent compound. The polyhistidine tag allowsconvenient isolation of the protein in a single step by nickelchromatography.

[0116] Alternatively, the fluorescent compound, such as a fluorescentprotein substrate, need not be isolated from the host cells. This methodis particularly advantageous for the assaying for the presence of anactivity in situ.

[0117] Methods for Determining Whether a Sample Contains an Activity

[0118] The present invention includes methods for determining whether asample contains an activity using a fluorescent compound of the presentinvention. Depending on the type of activity to be determined, differentfluorescent compounds are to be used. For example, if a proteaseactivity is to be determined, then a fluorescent compound that is asubstrate for a protease is used in the present methods. Likewise, if aprotein kinase activity is to be determined, then a fluorescent compoundthat is a substrate for a protein kinase is used in the presentinvention.

[0119] The present method for determining whether a sample contains anactivity comprises contacting a sample with a fluorescent compound ofthe present invention, wherein said fluorescent compound exhibitsquenching. The sample is contacted with a quenching agent, excited, andthe amount of emission from the fluorescent compound under quenchingconditions is measured.

[0120] As is known in the art, different cofactors are required fordifferent enzyme reactions. Thus, the skilled artisan would realize thatsuch cofactors should be present in the assay conditions for thoseenzymes. For example, protein kinases add a phosphate residue to thephosphorylation site of a protein generally through the hydrolysis ofATP to ADP. Fluorescent compounds that are substrates for proteinkinases are useful in assays to determine the amount of protein kinaseactivity in a sample. The assays of this invention take advantage of thefact that phosphorylation of the protein results in a change in afluorescent property of the fluorescent compound that can be detected byquenching. Methods for determining whether a sample has kinase activityinvolve contacting the sample with a fluorescent compound having aphosphorylation site recognized by the protein kinase to be assayed andwith a phosphate donor under selected test conditions. A phosphate donoris a compound containing a phosphate moiety that the kinase is able touse to phosphorylate the protein substrate. ATP(adenosine-5′-triphosphate) is by far the most common phosphate donor.In certain instances (such as whole cell studies) the sample willcontain enough of a phosphate donor to make this step unnecessary. Thenthe fluorescent compound is excited with light in its excitationspectrum in the presence and absence of at least one quenching agent. Ifthe fluorescent compound has been phosphorylated, the substrate willexhibit resistance to quenching, indicating that the sample containsprotein kinase activity. These general methods can be used to detect anyactivity using fluorescent compound, assay conditions, and quenchingconditions appropriate for an activity.

[0121] The amount of an activity in a sample can be determined bymeasuring the amount of quenching in the sample at a first time and asecond time after contact between the sample and the fluorescent proteincompound and determining the degree of change or the rate of change in aquenching. These results can be compared to those obtained with acontrol sample that does not contain the activity, or contains a knownamount of activity. The amount of an activity in the sample can becalculated as a function of the difference in the determined amount ofquenching at the two times. For example, the absolute amount of anactivity can be calibrated using standards of activity determined forcertain amounts of activity after certain amounts of time. The faster orlarger the difference in the amount of quenching, the more activity ispresent in the sample. The skilled artisan would realize that propercontrols should be used to validate any comparisons made with dataobtained from the sample.

[0122] Fluorescence in a sample is measured using a fluorimeter. Ingeneral, excitation radiation from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscauses the excitation radiation to excite the sample. In response,fluorescent compounds in the sample emit radiation that has a wavelengththat is different from the excitation wavelength. Collection optics thencollect the emission from the sample. The device can include atemperature controller to maintain the sample at a specific temperaturewhile it is being scanned. According to one embodiment, a multi-axistranslation stage moves a microtiter plate holding a plurality ofsamples in order to position different wells to be exposed. Themulti-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation. This process can be miniaturized and automatedto enable screening many thousand of compounds.

[0123] For example, comparisons can be made with a control sample knownnot to contain an activity, a control sample known to contain anactivity (preferably in a known amount), a control sample representingbackground signal, or a control sample with or without test compounds.

[0124] The sample can be any sample, such as a sample of cells, tissue,organ, or fluid obtained from an organism (such as a mammalian, such asa human) or an extract obtained therefrom. Miniaturized arrays ofsamples attached to a matrix, such as a bead or solid support as theyare known in the art or later developed, can be used in the presentinvention to detect fluorescence or other activity in a sample. A samplecan also comprise cultured cells, culture fluid, or extracts orconditioned media obtained therefrom. The cells can be prokaryotic oreukaryotic, such as mammalian cells, such as human cells.

[0125] Methods of performing assays on fluorescent materials are wellknown in the art. (Lakowics, Principles of Fluorescence Spectroscopy,Plenum Press, NY (1983); Herman, Fluorescence Microscopy of Living Cellsin Culture, Part B, Methods in Cell Biology, volume 30, Academic Press,San Diego, pp. 219-243 (1989); Turro, Modern Molecular Photochemistry,Menlo Park, Calif., Benjamin/Cummings Publishing, pp. 296-361 (1978)).

[0126] In one embodiment, a cell is transiently or stably transfectedwith an expression vector encoding a fluorescent compound containing asubstrate site for an activity to be assayed. This expression vectoroptionally includes controlling nucleotide sequences such as promoter orenhancing elements. The expression vector expresses the fluorescentcompound that contains the substrate site for an activity to bedetected. The activity to be assayed may either be intrinsic to the cellor may be introduced by stable transfection or transient co-transfectionwith another expression vector encoding the activity and optionallyincluding controlling nucleotide sequences such as promoter or enhancerelements. The fluorescent compound and the activity preferably arelocated in the same cellular compartment so that they have moreopportunity to come into contact. Membrane-bound or membrane-associatedfluorescent compounds can also be used in this and any other method ofthe present invention. The amount of activity is then determined bymeasuring the fluorescence of the sample (which can contain whole cells)under quenching conditions, and comparison to appropriate controls, suchas controls that either do not contain the activity, or contain a knownamount of activity.

[0127] A contemplated variation of the above assay is to use thecontrolling nucleotide sequence to produce a sudden increase in theexpression of either the fluorescent compound or the enzyme beingassayed, for example, by inducing expression of the construct.Fluorescence after quenching can be monitored at one or more timeintervals after the onset of increased expression. A large difference inthe amount of fluorescence after quenching over time reflects a largeamount or high efficiency of the activity. This kinetic determinationhas the advantage of minimizing any dependency of the assay on the basalor background levels of activity.

[0128] In another embodiment, the vector may be incorporated into anentire organism by standard transgenic or gene replacement techniques.An expression vector capable of expressing an activity optionally may beincorporated into the entire organism by standard transgenic or genereplacement techniques. Then, a sample from the organism containing thefluorescent compound is tested. For example, cell or tissue homogenates,individual cells, or samples of body fluids, such as blood, can betested.

[0129] Screening Assays

[0130] The methods of the invention can be used in drug screening todetermine whether a test compound alters an activity. In one embodiment,the assay is performed on a sample in vitro suspected of containing anactivity. A sample containing a known amount of activity is mixed with afluorescent compound of the invention and a test compound. The amount ofthe activity in the sample is then determined as above, for example bymeasuring the amount of fluorescence after quenching at a first andoptionally second time after contact between the sample, the fluorescentcompound, and the test compound and at least one quenching agent. Thenthe amount of activity in the presence of the test compound is comparedwith the activity in the absence of the test compound. A differenceindicates that the test compound alters the activity. The activity canbe increased, decreased, or unchanged by a test compound.

[0131] The present invention also includes a compound identified by anymethod of the present invention. Such compounds can be provided as apharmaceutical composition in a pharmaceutically acceptable carrier asis set forth in U.S. patent application Ser. No. 09/030,578, filed Feb.24, 1998. The present invention also includes a library of suchcompounds, which comprise two or more of such compounds provided eitherseparately or in combination. The present invention also includes asystem used to screen and identify compounds, such as set forth in U.S.patent application Ser. No. 08/858,016, filed May 16, 1997.

[0132] In another embodiment, the ability of a compound to alter anactivity in vivo is determined. In an in vivo assay, cells transfectedwith an expression vector encoding a fluorescent compound, such as afluorescent protein, of the invention are exposed to at least one amountof at least one test compound, and the fluorescence after quenching ineach cell (individually or as a population) can be determined.Typically, the difference is calibrated against standard measurements(for example, in the presence or absence of test compounds) to yield anabsolute amount of activity. A test compound that inhibits or blocks theactivity or expression of an activity can be detected by a relativechange in fluorescence after quenching. The cell can also be tranfectedwith an expression vector to coexpress the activity or an upstreamsignaling component such as a receptor, and the fluorescent compound.This method is useful for detecting signaling to an activity such as aprotein kinase of interest from an upstream component of a signalingpathway. If a signal from an upstream molecule, for example a receptor(preferably in the presence of an agonist), is inhibited by a testcompound, then the kinase activity will be inhibited as compared tocontrols incubated without the test compound. This provides a method forscreening for compounds that affect cellular events (includingreceptor-ligand binding, protein-protein interaction, or kinaseactivation), and signal to the target kinase. This method can usecultured cells or extracts or conditioned media derived therefrom. Thismethod can also use cells derived from an organism, such as a mammal,such as a human. Such cells can be derived from a tissue, organ orfluid. The sample can also comprise an extract of such cells.

[0133] This invention also provides kits containing a fluorescentcompound and optionally cofactors for an activity. In one embodiment,the kit comprises at least one container holding the fluorescentcompound and optionally a second container holding a cofactor or buffer.Optionally, the kit can comprise other reagents or labware to practice amethod, such as a method of the present invention. The entire kit can beprovided in a separate container, such as a box. This container caninclude instructions for use of the contents in a method of the presentinvention, or for other purposes.

[0134] Libraries of Candidate Substrates

[0135] This invention provides libraries of fluorescent compounds usefulfor the identification and characterization of sequences that can berecognized by an activity. Libraries of these fluorescent compounds canbe screened to identify sequences that can be modified by activities ofunknown or known substrate specificity, or to characterize differencesin activity in, or from, diseased and normal cells, tissues, fluids, orextracts thereof.

[0136] As used herein, a “library” refers to a collection containing atleast two different members, preferably greater than ten differentmembers. Each member of a fluorescent compound library comprises afluorescent protein moiety and a variable substrate site for anactivity, wherein the variable substrate site for an activity ispreferably located at or near the amino- or carboxy-terminus of thefluorescent protein moiety and has fewer than about fifty amino acids,preferably less than about fifteen amino acids. The variety of aminoacid sequences for the fluorescent protein moiety is at the discretionof the artisan. For example, the library can contain a diversecollection of variable peptide moieties in which most or all of theamino acid positions are subjected to a non-zero but low probability ofsubstitution. Also, the library can contain variable peptide moietieshaving an amino acid sequence in which only a few (e.g. one to ten)amino acid positions are varied, but the probability of substitution ateach positions is relatively high.

[0137] Preferably, libraries of fluorescent compounds are created byexpressing protein from libraries of recombinant nucleic acid moleculeshaving expression control sequences operatively linked to nucleic acidsequences that code for the expression of different fluorescentcompounds. Methods of making nucleic acid molecules encoding a diversecollection of peptides are described (U.S. Pat. No. 5,432,018 to Doweret al., U.S. Pat. No. 5,223,4098 to Ladner et al., U.S. Pat. No.5,264,563 to Huse et al., and WO 92/06176 to Huse et al.) For expressionof fluorescent compounds, recombinant nucleic acid molecules are used totranfect cells, such that each cell contains a member of the library.This produces, in turn, a library of host cells capable of expressingthe library of different fluorescent compounds. The library of hostcells can be used in the screening methods of this invention to identifyfluorescent compounds comprising a substrate site for an activity.

[0138] In one method of creating such a library, a diverse collection ofoligonucleotides having preferably random codon sequences are combinedto create polynucleotides encoding peptides having a desired number ofamino acids. The oligonucleotides preferably are prepared by chemicalsynthesis. The polynucleotides encoding the variable peptide moietiescan be coupled to the 5′ end of a nucleic acid coding for the expressionof a fluorescent compound or a carboxy-or amino-terminal portion of it.This is, the fluorescent protein moiety or a carboxy-terminal portion ofit. This creates a recombinant nucleic acid molecule coding for theexpression of a fluorescent compound having a peptide moiety fused tothe amino- or carboxy-terminus of a fluorescent protein moiety. Thisrecombinant nucleic acid molecule is then inserted into an expressionvector to create a recombinant nucleic acid molecule comprisingexpression control sequences operatively linked to the sequence encodingthe candidate substrate. The expression vector can then be inserted intoan appropriate cell and expressed. To generate the collection ofoligonucleotides which forms a series of codons encoding a randomcollection of amino acids and which is ultimately cloned into thevector, a codon motif is used, such as (NNK)x, where N may be A,C,G, orT (nominally equimolar, K is G or T (nominally equimolar), and x is thedesired number of amino acids in the peptide moiety, e.g. 15 to producea library of 1 5-mer peptides. The third positions may also be G or C,designated “S.” Thus, NNK or NNS (i) code for all the amino acids, (ii)code for only one stop codon, and (iii) reduce the range of codon biasfrom 6:1 to 3:1. The expression of peptides from randomly generatedmixtures of oligonucleotides in appropriate recombinant vectors isdiscussed in Olipant et al., Gene 44:177-183 (1986).

[0139] An exemplified codon motif (NNK)6 (SEQ ID NO:17) produces 32codons, one for each of 12 amino acids, two for each of five aminoacids, three for each of three amino acids and one (amber) stop codon.Although this motif produces a codon distribution as equitable asavailable with standard methods of oligonucleotide synthesis, it resultsin a bias against peptides containing one-codon residues.

[0140] An alternative approach to minimize the bias against one-codonresidues involves the synthesis of 20 activated tri-nucleotides, eachrepresenting the codon for one of the 20 genetically encoded aminoacids. These are synthesized by conventional means, removed from thesupport but maintaining the base and 5-OH-protecing groups, andactivating by the addition of 3′O-phosphoramidite (and phosphateprotection with beta-cyanoethyl groups) by the method used for theactivation of mononucleosides, as generally described in McBride andCaruthers, Tetrahedron Letters 22:245 (1983). Degenerate “oligocodons”are prepared using these trimers and building blocks. The trimers aremixed at the desired molar rations and installed in the synthesizer. Theratios will usually be approximately equimolar, but may be controlledunequal ratios to obtain the over- to under-representation of certainamino acids coded for by the degenerate oligonucleotide collection. Thecondensation of the trimers to form the oligocodons is done essentiallyas described for conventional synthesis employing activatedmononucleosides as building blocks (Atkinson and Smith, OligonucleotideSynthesis, Gait ed. pp. 35-82 (1984). Thus, this procedure generates apopulation of oligonucleotides for cloning that is capable of encodingan equal distribution (or a controlled unequal distribution) of thepossible peptide sequences.

[0141] Methods for Screening for Quenching Agents

[0142] The present invention includes methods for screening for agentsthat are quenching agents for a fluorescent compound of the presentinvention. As set forth in the Examples, fluorescent compounds that havea substrate site for an activity can exhibit quenching in the presenceof a quenching agent. Preferable quenching agents and quenchingconditions for a particular fluorescent compound can be followed byscreening a plurality of quenching agents and quenching conditionsfollowing the methods set forth in the Examples. For example, a firstsample of a fluorescent compound can be contacted with a control bufferand a second sample of a fluorescent compound can be contacted with anactivity. These samples can be incubated for any period of time, such asbetween about one minute and 72 hours, preferably between 1 and 6 hours.Aliquots of these samples can then be contacted with test quenchingagents and/or test quenching conditions, after which the fluorescence ofthese samples are measured. Samples that exhibit quenching can bereadily identified by measuring the appropriate fluorescence from thesamples (preferably by comparison with an appropriate control) whichidentify preferable quenching agents or quenching conditions. Morepreferable quenching agents or quenching conditions can be identified byreiterating this procedure using different concentrations of identifiedquenching agents for longer or shorter periods of time.

[0143] Methods for Screening Libraries of Candidate Substrates.

[0144] Libraries of host cells expressing fluorescent compounds areuseful in identifying fluorescent proteins having peptide moieties thatexhibit quenching. Several methods of using the libraries areenvisioned. In general, one begins with a library of recombinant hostcells, each of which expresses a different fluorescent compound, such asfluorescent compound comprising a protein, peptide, or nucleic acid.Each cell is expanded into a clonal population that is geneticallyhomogeneous.

[0145] In a first method, fluorescence quenching is measured or comparedfrom each clonal population before and after at least one specified timeafter a known change in an intracellular activity. Alternatively,fluorescence quenching measured in each clonal population can becompared with the results obtained using untreated control cells. Forexample, a change in kinase activity could be produced by transfectionwith a gene encoding a kinase activity, by increasing the expression ofthe kinase using expression control elements, or by any condition thatpost-translationally modulates the kinase activity. Examples of thelatter include cell surface receptor mediated elevation of intracellularcAMP to activate cAMP-dependent kinases, surface receptor mediatedincreases of intracellular cGMP to activate cGMP-dependent proteinkinase, increases in cytosolic free calcium to activateCa²⁺/calmodulin-dependent protein kinase types I, II, or IV, or theproduction of diacylglycerol to activate protein kinase C, etc. One thenselects for the clone(s) that show the largest or fastest changes influorescence in response to quenching compared to non-treated controlcells.

EXAMPLES

[0146] A. Phosphorylation Sites Located in the Amino Acid Sequence ofAequorea GFP Remote in the Primary Amino Acid Sequence Form theN-Terminus

[0147] Potential sites for phosphorylation were chosen at or close topositions in GFP that had previously been identified on the basis ofmutagenesis experiments to exert significant effects on fluorescence, orwhich had a higher probability of surface exposure based on computeralgorithms. For example, in mutant H9, Ser 202 and Thr 203 are mutatedto Phe and lie, respectively, creating a large change in spectralproperties. Therefore, in one mutant. 199 RRLSI (SEQ ID NO:18), apotential site of phosphorylation was created around Ser 202, whosephosphorylation would significantly affect the fluorescent properties ofthe parent molecule. Similarly, the amino acids located at positions72and 175 have been implicated in increased folding efficiency of GFP athigher temperatures and were made into potential sites ofphosphorylation in separate mutants.

[0148] A complete list of the positions and amino acid changes made foreach phosphorylation mutant in this series is outlined in FIG. 2.Proteins were expressed in E. coli using the expression plasmid pRSET(Invitrogen, CA), in which the regions encoding GFP was fused in framewith nucleotides encoding an N-terminal polyhistidine tag (FIG. 3). Thesequence changes were introduced by site-directed mutagenesis using theBio-Rad mutagenesis kit (Kunkel, Proc. Natl. Acad. Sci. 82:488-492(1985)); and Kunkel et al., Meth. Enzymol. 154:367-382 (1987)) andconfirmed by sequencing. The recombinant proteins were induced with 0.05mM IPTG, expressed in bacteria and purified by nickel affinitychromatography. The sequence changes, relative fluorescence, relativerater of phosphorylation and the percent change in fluorescence uponphosphorylation are listed in Table IV. In those cases where the proteinexhibited no fluorescence after insertion of the phosphorylation site,no determinations were made on the effect of phosphorylation onfluorescence. TABLE IV Relative fluorescence, rate of phosphorylationand change in fluorescence upon phosphorylation for mutantsincorporating phosphorylation sites remote from the N- terminus. %change in Fluorescence before fluorescence after phosphorylation (%Relative rates of incubation with SEQ ID NO: Sequence of wild type)physphorylation kinase SEQ ID NO.19 25RRFSV 95 1.75 −5 SEQ ID NO.2068RRFSR 0 N.D. N.D. SEQ ID NO.14 68RRFSA 6 0.6 10 SEQ ID NO.21 94RRSIF 0N.D. N.D. SEQ ID NO.22 131RRGSIL 0 N.D. N.D. SEQ ID NO.23 155KRKSGI 862.5 0 SEQ ID NO.24 172RRGSV 90 1.57 0 SEQ ID NO.18 199RRLSI 0 N.D. N.D.SEQ ID NO.15 214KRDSM 21 1.88 40

[0149] Numbers prior to the sequence indicate amino acid position inwild type GFP (SEQ ID NO:2) where phosphorylation motif starts. Therelative rates of phosphorylation compare the rate of phosphorylation ofthe given phosphorylation motif with the endogenous protein kinase Aphosphorylation motif in Aequorea GFP (HKFSV. SEQ ID NO:1) measured byincorporation of ³²P after incubation of the purified substrate andprotein kinase A catalytic subunit in the presence of ³²P-labeled ATPusing 3 micrograms of GFP, 5 micrograms protein kinase A catalyticsubunit, for 10 minutes at 30° C. in standard phosphorylation buffer (20mM MOPS, pH 6.5, 100 mM KCl, 100 micromolar ATP, 3 mM MgC₂, 1 mM DTT and100 microCi ³²P-labeled ATP. Reactions were terminated by blotting ontophosphocellulose paper and washing with 10% phosphoric acid. The percentchange in fluorescence represents the increase in fluorescence (475 nmexcitation, 510 nm emission) observed in each purified protein resultingfrom incubation with excess protein kinase A catalytic subunit for onehour at 30° C. using the same phosphorylation conditions as describedabove except that no ³²P-labeled ATP was present and that after thereaction time was complete, samples were analyzed in a fluoromiterrather than blotted onto phosphocellulose paper.

[0150] The greatest change in fluorescence occurred in mutant 214KRDSM(SEQ ID NO:15) which exhibited at 40% change in fluorescence uponphophorylation. However. analysis of the kinetics of phosphorylationusing gamma-32P-labeled ATP demonstrated that the site is poorlyphosphorylated by protein kinase A. Wild type GFP contains a mediocreconsensus phosphorylation motif (25HKFSV, SEQ ID NO:1) that can bephosphorylated by protein kinase A in vitro with relatively slowkinetics. While phosphorylation at this position has no detectableeffect on the fluorescence of GFP, the rate of phosphorylation at thisposition is used as an internal control between experiments to determinethe relative rates of phosphorylation at sites engineered into theprotein by site directed mutagenesis.

[0151] B. Phosphorylation Sites At or About the N-Terminus of AequoreaGFP

[0152] Phosphorylation sites at the N-terminus of GFP were engineeredinto S65T GFP by PCR. The sequence changes, relative fluorescence,relative rates of phosphorylation and the percent change in fluorescenceupon phophorylation are tabulated in Table V. TABLE V Relativefluorescence, rate of phosphorylation and change in fluorescence uponphosphorylation for phosphorylation sites inserted at the N-terminusRelative fluorescence as a Relative rates of % Change in SEQ ID NO:Sequence % of wild type phosphorylation fluorescence SEQ ID NO.21MSKGEELF 100 1.0 0 SEQ ID NO.25 1MRKGSCLF 40 5.1 5.7 SEQ ID NO.261MRKGSLLF 52 1.6 8.0 SEQ ID NO.27 1MRRESLLF 30 3.0 6.0 SEQ ID NO.281MRDSCLF 27 3.7 17 SEQ ID NO.29 1MSRRDSCF 43 2.1 25 SEQ ID NO.301MSKRRDSL 7 5.5 5.1

[0153] Numbers prior to the sequence indicate amino acid position in thewild type GFP where the phosphorylation motif starts. The relative ratesof phosphorylation compare the rate of phosphorylation of the givenphosphorylation motif with the endogenous protein kinase Aphosphorylation motif in Aequorea GFP (HKFSV) measured by incorporationof ³²P after incubation of the purified substrate and protein kinase Acatalytic subunit in the presence of ³²P-labeled ATP using the standardprotocols described above. The percentage change in fluorescencerepresents the change in fluorescence (488 nm excitation, 511 nmemission) observed in each purified protein as a result of incubationwith excess protein kinase A catalytic subunit for one hour at 30° C.using phosphorylation conditions described above. These resultsdemonstrate that mutants whose sequence closely resembles the nativeprotein retain considerable fluorescence, display good kinetics ofphosphorylation, but show relatively small changes in fluorescence afterphosphorylation. To improve the effect of phosphorylation onfluorescence, amino acids around the phosphorylation site were mutatedto create an optimal phosphorylation sequence even if it disordered theexisting local tertiary structure. Such disruption was predicted andfound to decrease the basal fluorescence of these constructs in theirnon-phosphorylated state (Table VI). TABLE VI Relative fluorescencebefore phosphorylation and change in fluorescence upon phosphorylationfor more drastically altered phosphorylation sites inserted at theN-terminus. Relative % Change in fluor- fluor- escence escence as a % ofGFP upon mutant phosphory- SEQ ID NO: Sequence S65T lation SEQ ID NO.21MSKGEELF 100  0 (WILD TYPE) SEQ ID NO.31 1MSRRRSI 5.8 40 SEQ ID NO.321MRRRRSII 5.1 70 SEQ ID NO.33 −1MRRRRSIII N.D. 43 SEQ ID NO.34−2MRRRRSIIIF 0.7 15 SEQ ID NO.35 −3MRRRRSIIIIF 0.6 70

[0154] Numbers prior to the sequence indicate amino acid position inwild type GFP where the phosphorylation site starts. Negative numbersindicate extensions onto the wild-type N-terminus. The percent change influorescence represents the change in fluorescence (488 nm excitation,51 1 nm emission) observed in each purified protein resulting fromincubation with excess protein kinase A catalytic subunit for one hourat 30° C. using standard phosphorylation conditions described earlier.

[0155] Perhaps because of the reduced basal fluorescence,phosphorylation by protein kinase A produced greater percentageincreases in fluorescence in these constructs than in the moreconservative mutations of Table IV. Constructs 1MRRRRSII (SEQ ID NO:32),MRRRRSIII (SEQ ID NO:33) and −3MRRRRSIIIIF (SEQ ID NO:35) displayed thegreatest increases, about 70%, in fluorescence upon phosphorylationusing the standard phosphorylation conditions. However, these increasedpercentage increases were obtained at the cost of reduced ability tofold at higher temperatures and relatively poor fluorescence even afterphosphorylation. To improve these characteristics, these mutants werefurther optimized by additional random mutagenesis with a novelselection procedure.

[0156] C. Further Optimization of N-Terminal Phosphorylation Sites byRandom Mutagenesis of the Remainder of GFP

[0157] The two best constructs from above (1MRRRRSII (SEQ ID NO:32) and−3MRRRRSIIIIF (SEQ ID NO:35)) were further mutagenized and screened forvariants that were highly fluorescent when phosphorylated, but weaklyfluorescent when non-phosphorylated. The method involved expression of arandomly mutated fluorescent compound with or without simultaneousco-expression of the constitutively active catalytic subunit of proteinkinase A in bacteria, and screening the individual mutants to determinethose fluorescent compounds that are highly fluorescent in the presencebut not the absence of the kinase.

[0158] To enable co-expression of the kinase and fluorescent compoundsuch as GFP, a new expression vector with the kinase A catalytic subunitupstream from the fluorescent substrate was constructed (FIG. 4). Thisconstruct enabled expression of both the kinase and GFP from the samepromoter through the insertion of a ribosome-binding site between thecoding regions of the first and second genes. Random mutations wereintroduced into GFP by error-prone PCR and the resulting population ofmutants cloned into the co-expression vector using the appropriaterestriction sites. The expression library of vectors contained themutated fluorescent compounds were transformed into host bacteria andindividual bacterial colonies (each derived from a single cell, andhence containing a single unique mutant fluorescent compound) werecultured.

[0159] The colonies were screened for fluorescence either byfluorescence-activated cell sorting or by observation of individualcolonies grown on an agar plate under a microscope. Those colonies thatexhibited the greatest fluorescence were re-screened under conditions inwhich the kinase gene was inactivated. This was achieved in either oftwo ways. In the first method the co-expression vector was isolated andtreated with restriction endonucleases and modifying enzymes (EcoR1,klenow fragment, and T4 DNA ligase) to cut the kinase gene, addadditional bases and religate the DNA, causing a frame shift and henceinactivating the gene. The treated and non-treated plasmids were thenre-transformed into bacteria and compared in fluorescence.Alternatively, the plasmids were initially grown in a RecA− (recombinaseA negative) bacterial strain. where the kinase is stable, to screen forbrighter mutants in the presence of the kinase. The plasmid DNA was thenisolated and re-transformed into a strain of bacteria which is RecA+. inwhich the kinase is unstable and is lost through homologousrecombination of the tandomly repeated ribosome biding sites (rbs). Thebacteria have a strong tendency to eliminate the kinase A catalyticsubunit because it slows their multiplication, so cells that splice outthe kinase by recombination have a large growth advantage.

[0160] Comparison of the brightness of the mutant first in the presenceof kinase then in its absence indicates the relative effect ofphosphorylation on the mutant GFP fluorescence (after normalizing forGFP expression levels). A library of approximately 2×10⁶ members wasscreened by this approach. Approximately 500 mutants displayed higherlevels of fluorescence when screened in the presence of the kinase.After inactivation of the kinase, one mutant out of the 500 displayedreduced levels of fluorescence. The increased fluorescence of theremainder of the 500 mutants was independent of the presence of thekinase. This mutant GFP was isolated and sequenced and found to containthe following mutations compared to wild-type GFP (SEQ ID NO:2) (inaddition to the N-terminal phosphorylation site 1MRRRRSII (SEQ IDNO:32)): S65A, N149K, V163A and 1167T).

[0161] To confirm that this mutant was indeed directly sensitive toprotein kinase A phosphorylation and to quantify its responsively, itwas expressed in the absence of kinase. The E. coli were lysed and theprotein purified as described earlier using a nickel affinity column.The protein exhibited high levels of fluorescence when induced at 30° C.but displayed reduced fluorescence when incubated at 37° C. After suchpreincubation (37° C. overnight) and separation of the less fluorescentmaterial by centrifugation, this protein exhibited the largest change influorescence upon phosphorylation yet observed. The tolerance of thismutant for 37° C. treatment suggested that this mutant is suitable foruse in mammalian cells.

[0162] D. GFP Mutants Exhibiting Phosphorylation Dependent Quenching

[0163] A phosphorylation recognition motif and substrate site forprotein kinase A was engineered into the N-terminal region of GFP havingthe mutations S65A. N149K. V163A, and 1167T (Examples A to C). Furthermutations were made within the coding sequence of GFP at positions thatwere identified to be in close three-dimensional contact with the siteof phosphorylation (phosphorylation site is at Glu5 in the wild typeprotein (SEQ ID NO:2)). These mutants were designed to strengthen ionicinteractions between the phosphoserine and internal positively chargedamino acids such as Lvs79, for example by mutation of Lys79 to Arg orHis. Additional mutations were also made to disorder the localN-terminal structure of the GFP in the non-phosphorylated form, forexample, by disrupting the interactions between Lys3 and Glu90, bymutation of Glu90 to Lys or Asn. These mutations were made to bothenhance the effect of phosphorylation on the fluorescent properties ofGFP and to improve the accessibility of the phosphorylation motif orsite to the kinase.

[0164] Mutation of amino acids close in sequence to the site ofphosphorylation can also be changed to further weaken their interactionswith other amino acid residues, although the sequence around the site ofphosphorylation may directly impact the efficiency of phosphorylation byaltering or disrupting the recognition motif for phosphorylation. Anexample of such a change is the mutation of Phe8 to the smaller and lesshydrophobic amino acid Leu, which can disrupt or reduce hydrophobicinteractions between Phe8 and LysS5, Cys70, Leu 19 and Met88. Also,mutation of Gly4 to Ala provides a relatively small hydrophobic aminoacid that is preferred as a phosphorylation motif, and would not distortthe interaction between the point of phosphorylation and its point ofinteraction within the GFP molecule. Not wishing to be bound to anymechanism of action, the inventors postulate that the phosphorylation ofGFP may result in a transition from a locally disordered to orderedstate without initially causing gross changes in protein conformation.This change can cause different fluorescent properties of the GFP in thephosphorylated and non-phosphorylated states under quenching conditions.

[0165] GFP mutant K4 (SEQ ID NO:49) (−2M,−1G,M1R,S2R,K3R,G4A,E5S,E6I,L7I,S65A,N149K,V163A,I167T) which contains aprotein kinase A substrate recognition motif and substrate site for anactivity, was used as the basis for other mutants K5 to K16. Singlemutants K79R (K5), E90N (K6), E90K (K7) and double mutants K79R/E90N(K8), K79R/E90K (K9), K79H, E90N (K10), K79H, E90K (K11), K79H (K12),K79E, E90N (K13), K79E, E90K (K14), K79E (K15) and K79Q (K16) of K4 weremade using known methods (see, Sambrook, Molecular Cloning. A LaboratoryManual, Cold Spring Harbor Press (1989)).

[0166] 1. Use of Low pH as a Quenching Agent to Enhance FluorescenceChanges of GFP Mutants Upon Phosphorylation

[0167] Mutants K4, K5, K6, K7, K8 and K9 were evaluated for fluorescenceproperties in their phosphorylated and non-phosphorylated states as afunction of pH. Individual GFP mutants (4 micromolar) werephosphorylated by incubation with mouse recombinant protein kinase Acatalytic subunit (Calbiochem #539-487, specific activity of 7,100 unitper milligram of protein) (1 unit in 20 mM MOPS, pH 7.3, 1 mM DTT, 3 mMMgCl₂, 1 mM ATP at 30° C. for 1 hour in a total volume of 50microliters). Control samples were incubated under the same conditionswithout protein kinase A. All fluorescence measurements were made usingthe Perseptive Biosystems 96 well plate reader with standard excitationand emission filters (Ex 485/25. Em 530/30) and gain setting of 70.Measurements were made approximately five minutes after addition of 100microliters of the quenching buffer (50 mM citrate, 100 mM NaCl)provided at the indicated pH. The results represent the means oftriplicate determinations. The fluorescence of the phosphorylated GFPmutant relative to the non-phosphorylated GFP mutant was calculated andpresented in Table VII. These data demonstrate that mutants can exhibitchanges in a fluorescent property upon quenching at low pH. and thattheir sensitivity to quenching is different for different mutants. TABLEVII Effect of pH on Quenching of GFP Mutants Fold Change in Fluorescenceof GFP in the Phosphorylated State at the Indicated pH Compared toNon-Phophorylated Samples Mutant 5.6 5.4 5.2 5.0 4.8 K4 0.84 0.86 0.911.04 1.92 K5 1.0 1.0 1.05 1.6 1.9 K6 1.0 1.0 1.0 1.6 2.5 K7 0.95 0.951.0 1.4 2.0 K8 0.90 0.90 1.0 1.4 2.8 K9 1.0 1.0 1.0 1.3 2.3

[0168] The composition of the buffer used to stabilize the pH at theindicated value (for example, acetate, or citrate/phosphate) had littleeffect on quenching. Acetate buffers provided slightly greater and morerobust changes than citrate. The highest degree of quenching in thisexample occurred using 100 mM acetate buffer in the presence of 100 mMNaCl at a volume that was twice that of the sample (standard quenchingconditions). Preferred quenching conditions were dependent on the samplepH and the GFP mutant.

[0169] 2. Effect of Time of Incubation and Low pH on Fluorescence ofPhosphorylated and Non-Phosphorylated GFP mutant K8

[0170] The time dependency of changes in quenching were investigatedusing GFP mutant K8 over a range of pH values using standard quenchingbuffer (Table VIII). The procedures described for the data presented inTable VII were used for these experiments, except that measurements weremade at the indicated times. The “Time of Incubation” column representsthe amount of time that the samples were under quenching conditionsbefore fluorescence measurements were taken. TABLE VIII Effects of Timeon Quenching of GFP Mutant K8 Fold Increase in Time of IncubationFluorescence of GFP in (hours) Optimal pH the Phosphorylated Sate 0 4.61.5 0.5 4.8 3.3 1.5 5.0 3.7 4.5 5.0 5.1 7.5 5.0 6.1 10.5 5.0 7.5

[0171] Lower pH values of the quenching agent (for example, pH 4.6 orbelow) resulted in relatively smaller changes in fluorescence comparedto control samples that were maximal relatively rapidly after additionof the quenching agent. Higher pH 1 values (4.8 to 5.0) of the quenchingagent resulted in larger differences in fluorescence, although thesechanges required larger times of incubation (up to ten hours). Maximaleffects of quenching with low pH buffers were obtained around pH 5.0+0.2with 100 mM acetate buffer with 100 mM NaCl. Maximal effects of low pHquenching were obtainable after ten to twenty-four hours, depending onthe pH of the quenching agent used. After this time, fluorescencedifferences were stable for up to 72 hours. If the pH of the quenchingagent (100 mM sodium acetate with 100 mM NaCl) was above pH 5.4,phosphorylation mediated fluorescence changes remained small, even up to24 hours of incubation These results are summarized in FIG. 5.

[0172] Quenching with low pH buffer caused a decrease in the relativefluorescence of the non-phosphorylated GFP mutant K8 compared to thephosphorylated GFP mutant K8 (see FIG. 6).

[0173] 3. Effect of Ionic Strength, Detergents, and Organic Solvents onFluorescence of Phosphorylated and Non-Phosphorylated GFP Mutants

[0174] The relative quenching of GFP mutant K8 in a phosphorylated andnon-phosphorylated state was enhanced by the presence of 100 mM NaCl.Higher or lower concentrations of salt reduced the magnitude andkinetics of quenching for both phosphorylated and non-phosphorylatedsamples, but did enhance the relative difference in quenching betweenphosphorylated and non-phosphorylated samples. The inclusion of adivalent cation chelator such as EDTA or CDTA stabilized thefluorescence of phosphorylated GFP mutant K8, possibly by inhibitingacid phosphatases present as a contaminant in the sample or buffer.Beta-glycerol phosphate (Sigma) (25 mM) was also an effective inhibitorof acid phosphatase activities.

[0175] The detergents Triton® X-100, Tween® 20. NP-40 and CHAPS® (in theconcentration range of 0-01 to 2 percent) in 100 mM acetate buffer, 100mM NaCl, pH 4.6 to 9.0 reduced the fluorescence of both thephosphorylated and non-phosphorylated samples and increased the rate ofloss of fluorescence of both the phosphorylated and non-phosphorylatedsamples. Based on these results, these quenching agents can be includedin the quenching conditions to accelerate the rate of quenching whichcan make these assays more convenient.

[0176] Urea and guanidine HCl (up to a concentration of 3 M) at pH 7.0did not significantly enhance the relative quenching of phosphorylatedor non-phosphorylated GFP mutant K8.

[0177] 4. Analysis of Phosphorylation Kinetics by Radiolabel-BasedMeasurements of Protein Phosphorylation

[0178] The phosphorylation kinetics of GFP mutant K8 (−2M, −1G, M1R,S2R, K3R, G4A, E5S, E61, L7I, S65A, K79R, E90N. N149K, V163A, 1167T) anda control which lacked the N-terminal phosphorylation motif and sitepresent in the mutant K8 were determined using the incorporation of³²P-phosphate (FIG. 7). Experiments were conducted using followingreaction conditions: 20 mM MOPS, pH 7.4, 100 mM KCl, 0.2% Tween® 20, 2.5units of protein kinase A. Phosphorylation reactions were initiated bythe addition of radiolabeled ATP and magnesium (5 μCi ³²P-ATP per tube)to GFP mutant K8 at a concentration of 2 micromolar. Phosphorylationreactions were performed for the indicated times at 30° C. and wereterminated by the addition of 10% trichloroacetic acid (TCA). Bovineserum albumin was added as a carrier (10 microliters of 1% BSA per tube)and the resulting precipitate was collected by centrifugation. Theresulting pellet was washed three times in 10% TCA prior to countingradioactivity by Cerenkov counting. The GFP mutant K8 exhibited greaterincorporation of ³²P than the control that lacks the N-terminalphosphorylation site. The results of these experiments demonstrate thatthe GFP mutant K8 is rapidly phosphorylated by protein kinase A.

[0179] Kinetic analysis of the rate of phosphorylation of GFP mutant K8having the phosphorylation motif set forth in SEQ ID NO:49 measured by³²P incorporation revealed an apparent Km of 9 μM and a turnover number(Kcat) of 1.9 sec⁻¹ at 30° C. Analysis of these parameters based onquenching alone gave an estimated Km of 7.7 μM and Kcat of 1.2 sec⁻¹.Thus, both of these methods gave similar results, validating the use ofquenching alone to determine these parameters and phosphorylation ingeneral.

[0180] 5. Validation of GFP Mutant K8 for Use in 96 Well HomogeneousFluorescence-Based Kinase Assays.

[0181] The limit of detectability of protein kinase A using theGFP-based fluorescent assay set forth in these examples in a 96 wellassay format was determined by measuring the fluorescence changes inresponse to incubation with a range of protein kinase A concentrations,as is described in Table VII. Reactions were performed under standardconditions for forty minutes at 30° C. Fluorescent measurements weremade using a Cytofluor 2 series 4000 from Perseptive Biosystem 96 wellplate reader fitted with standard excitation and emission filters(485/25, 530/30) and set at a gain of 70. Assays were performed usingCostar 96 well black plates with clear bottom in a reaction volume of 50microliters. Reactions were terminated by addition of the preferredacetate quenching conditions (100 mM acetate buffer pH 5.0, 100 mM NaCl,25 mM Beta-glycerol phosphate ).

[0182] At the lowest concentration of PKA tested (26 pmol, or 0.5 ng) adetectable change in fluorescence signal was observed upon quenching.Fifty-two pmol of PKA gave an approximately two-fold increase influorescence compared to controls that were incubated in the absence ofPKA. These results demonstrate that the assay provides highly sensitivemeasurements.

[0183] 6. Detecting GFP Mutant K8 Using 96-well plate reader.

[0184] These instrument settings and plates were used to determine thelimit of detection of GFP mutant K8. In TRIS buffer at pH 8.0, the GFPmutant K8 was detectable above background fluorescence at approximately0.1 μM. After treatment with the preferred quenching conditions at pH5.0 GFP mutant K8 was detectable above background fluorescence at alimiting concentration of about about 0.5 μM. These results demonstratethat the GFP mutant K8 can be detected with high sensitivity usingstandard 96-well plate readers in a typical screening environment.

[0185] 7. Robustness of the Assay to the Effects of Co-Solvents.

[0186] Assay robustness to co-solvents is a highly desirable feature ofdrug screening systems because many drug candidates are not appreciablysoluble in aqueous solutions. The co-solvents DMSO or ethanol arefrequently used in drug screening at concentrations up to about 1% todissolve drugs or target compounds. Thus, it is important to establishthat these agents alone do not significantly influence the quenching ofGFP at these concentrations. The preferred assay conditions as describedabove were used to determine the effects of the solvents DMSO andethanol on the GFP mutant K8 phosphorylation assay.

[0187] DMSO or ethanol were added at 0, 0.01, 0.1, 0.2, 0.5, and 10%(Vol/Vol) to the kinase reaction mixture. At the maximum concentrationstested, these agents exhibited little or no effect of phosphorylation onthe fluorescence development after quenching. These results establishthat co-solvents such as DMSO or ethanol at concentrations used inscreening assays do not interfere appreciably with GFP mutant K8quenching assays.

[0188] 8. Assay Validation with Protein Kinase A Inhibitors.

[0189] The protein kinase A inhibitors PKI (protein kinase A heat-stableinhibitor, isoform alpha (Calbiochem #539488) and H-89(N-[2-((p-bromocinrinamyl)amino)ethyl]-5-isoquinoline sulfonamide, HCl)(Calbiochem #371963) were tested in the preferred assay condition todetermine if they could be detected using the GFP mutant K8 quenchingassay. Both compounds were tested individually (between 0.1 and 1,000nM) and pre-incubated with PKA in the absence of mutant K8 for 10minutes at 4° C. Then, GFP mutant K8 was added to a final concentrationof 4 μM and the mixtures were incubated for thirty minutes at 30° C. Thekinase reactions were terminated by the addition of the preferredacetate quenching conditions (100 mM acetate, 100 mM NaCl. pH 5.0, 25 mMbeta-glycerol phosphate). The fluorescence of these samples was measured14 to 16 hours later. The results of these studies are presented in FIG.8. The results of these studies establish that the GFP mutant K8quenching assay can be used to detect kinase inhibitors and furthervalidates the methodology for drug screening.

[0190] 9. Detection of ATP Antagonists Using GFP Mutant K8 QuenchingAssay

[0191] The protein kinase A inhibitor H-89 was tested in the GFP mutantK8 kinase assay at two different ATP concentrations (0.1 mM and 1 mM).In the presence of the higher ATP concentration, the inhibitor was muchless efficient at inhibiting kinase activity. The response of theinhibitor to different ATP concentrations indicates that it acts byinhibiting ATP binding to the kinase active site. Because the GFP kinaseassay can be performed at both high and low ATP concentrations, thismethod can be used to identify, measure, and detect ATP antagonists.Furthermore assay conditions can be established (i.e. the use of high orlow ATP levels) to select for, or screen out, such compounds. Thisprovides a significant improvement over competing assay technologiessuch as radioactive incorporation that can only be run at highsensitivity with low ATP concentrations. The results of these studiesestablish that the GFP mutant K8 quenching assay can be used to detectkinase inhibitors and further validates the methodology.

[0192] 10. Use of GFP to Measure Other Kinase Activities.

[0193] In addition to GFP mutant K8 having a PKA phosphorylationrecognition motif, the inventors have made versions of GFP mutant K8that have phosphorylation recognition motifs that are selective forprotein kinase C (PKC) and mitogen activated protein kinase (MAP) (alsoknown as extracellular regulated kinase (erk) (Table IX)). Thesesubstrates have the same site of phosphorylation as GFP mutant K8, whichcorresponds to Glu5 in the wild-type protein. TABLE IX Additionalprotein phosphorylation motifs introduced into GEP mutant K8. RelativeFluorescence Compared to Clone GFP mutuant Kinase SpecifityPhosphorylation Motif Name K8 Corresponding wild-type Met Ser Lys GlyGlu Glu Len Phe (SEQ ID NO.36) Wild type sequence Erk Kinase Met Val GluPro Leu Thr Pro Ser Phe (SEQ ID NO.64) Erk-1 1.40 Erk Kinase Met Thr GlyPro Leu Ser Pro Gly Phe (SEQ ID NO.65) Erk-4 1.49 Erk Kinase Met Thr GlyPro Leu Ser Pro Gly Tyr (SEQ ID NO.66) Erk-5 1.26 Erk Kinase Met Thr GlyPro Leu Ser Pro Gly Leu (SEQ ID NO.67) Erk-6 1.22 Erk Kinase Met Thr GlyPro Leu Ser Pro Gly Pro (SEQ ID NO.68) Erk-7 0.40 PKC α Arg Arg Arg ArgArg Lys Gly Ser Phe Arg (SEQ ID NO.56) Pkc 3 0.72 PKC α (+Membrane ArgArg Arg Arg Arg Lys Gly Ser Phe Arg (SEQ ID NO.57) Pkc 3 lys 0.95association motif (hepta- Lys) at the C-terminus)) PKC β1 Phe Lys LeuLys Arg Lys Gly Ser Phe Lys (SEQ ID NO.58) Pkc 4 1.1.3 PKC δ Ala Arg ArgLys Arg Lys Gly Ser Phe Phe (SEQ ID NO.59) Pkc 5 1.91 PKC ε Tyr Tyr AlaLys Arg Lys Met Ser Phe Phe (SEQ ID NO.60) Pck 6 1.09 PKC ζ Arg Arg PheLys Arg Gln Gly Ser Phe Phe (SEQ ID NO.61) Pkc 7 1.88 PKC μ Ala Ala LeuVal Arg Gln Met Ser Val Ala (SEQ ID NO.62) Pkc 8 1.84

[0194] MAPKs (for mitogen-activated protein kinases) or ERKs (forextracellular-regulated kinases) selective phosphorylation motifs wereintroduced into GFP mutant K8 based on their known preferred substraterecognition motifs. Phosphorylation motifs were designed based onSongyang et al., Mo. Cell Biol. 16:6486-6493 (1996) and were made byreplacing the protein kinase A phosphorylation motif in the GFP mutantK8 with the indicated phosphorylation motif using PCR methods known inthe art. PKC isoform specific phosphorylation motifs were based on thesequences identified by Nishikawa et al. J. Biol. Chem. 272:952-960(1997). These phosphorylation motifs were introduced into GFP mutant K8by PCR as described above.

[0195] All of the constructs were successfully expressed at high leveland were highly fluorescent (Table IX). The ERK substrates showed nosubstantial sequence identity with either the wild-type GFP or the PKAmotifs present in GFP mutant K8, yet in most cases were as fluorescentor more fluorescent than GFP mutant K8. These results demonstrate thatmany different N-terminal phosphorylation motifs can be successfullyintroduced into GFP without significantly impacting GFP fluorescence.

[0196] E. Rates and Efficiencies of Phosphorylation of Additional GFPSubstrates

[0197] 1. Erk Selective Substrates

[0198] Table X reports the phosphorylation rates of various GFP mutantsensors containing Erk selective recognition motifs. The constructsErk-6 and Erk-7 exhibited the greatest rates of phosphorylation. Kineticanalysis of the Erk-7 construct revealed a Km of 15 μM and a Kcat of0.055 sec⁻¹. TABLE X Phosphorylation rates of various GFP Kinase SensorsSample ³²P-Incorporation (CPM) Control 102 ± 32 Myelin Basic Protein33361 ± 477  Erk-1 1579 ± 282 Erk-4 1982 ± 260 Erk-5 6455 ± 558 Erk-622498 ± 381  Erk-6-B17 13,499 ± 472   Erk-7 25502 ± 2077

[0199] These studies were performed by incubating GFP or MBP (myelinbasic protein) (10 μM) with activated recombinant MAP kinase (Calbiochem#454855) (100 ng) for 30 minutes at 30° C. in buffer (20 mM MOPs, pH7.2, 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM DTT, 0.1 mM ATP, 20 mMMgCl₂ 10 μCi ³²P-ATP in a volume of 50 microliters). Experiments wereterminated and samples processed as described previously in subsectionD4. Results presented in Table X represent means of triplicatedeterminations±standard deviations of ³²P-incorporation in washedpellets as described earlier.

[0200] The value of Kcat obtained for the GFP Erk substrate was similarto the Kcat values obtained for myelin basic protein, awell-characterized substrate of Erk kinase. These results demonstratethat Erk-1 selective phosphorylation motifs can be introduced into GFPand that the site of phosphorylation is rapidly and efficientlyphosphorylated, with comparable kinetics to other proteins or peptidesthat are known substrates of Erk-1 kinases.

[0201] a. Mutagenesis of Erk Selective Substrates Erk-6 and Erk-7 toImprove Fluorescence and Kinetics of Phosphorylation.

[0202] To improve the fluorescent properties of the Erk kinasesubstrates (for example, Erk-6 and Erk-7), a library of mutants derivedfrom these clones was made in which amino acids in the interior of theGFP that interact (with the three-dimensional structure of the protein)with the N-terminal region of GFP were mutated. The mutants weredesigned to produce a “better fit” of the Erk phosphorylation motif intothe top of the barrel of GFP. This was achieved by enhancing the size ofthe positive charge associated around the site of phosphorylation (bymutation of K85 to R), pushing the backbone amide chain closer to theN-terminal phosphorylation recognition motif (by mutation of A87 tolarger amino acids) and by making E90 more hydrophobic so that it couldattract Pro 3 and therefore move closer to the phosphorylation motif.This library of mutants were screened for improved brightness andfolding. Mutagenesis resulted in the creation of better folding, and amore fluorescent version, of the Erk-6 mutant, but did not significantlyimprove the fluorescence or folding of the Erk-7 mutant.

[0203] Selected clones from the Erk-6 and Erk-7 mutagenesis reactionswere sequenced to confirmed that mutagenesis was successfullyaccomplished. The non-wild type sequences are displayed in TABLE XI.Therein, poorly fluorescent clones exhibited less than 10% of thefluorescence of GFP mutant K8 and were not further characterized. TheGFP mutant Erk-6-B 17 showed 158% of the fluorescence of GFP mutant K8,demonstrating that the mutagenesis approach was successful in improvingGFP fluorescence. TABLE XI Mutants of Erk-6 and Erk-7 Substrates. MutantName Mutations Fluorescence E6-B17 A87T, E90A Highly fluorescent E6-A5K85R Poorly Fluorescent E6-A10 K85R, A87T, E90L Poorly FluorescentE6-A14 K85R, A87V, E90P Poorly Fluorescent E6-A17 K85R, A87T, E90SPoorly Fluorescent E7-B19 E901 Poorly Fluorescent E7-B22 A87T, E90RPoorly Fluorescent E7-A40 K85R, A87T, E90N Poorly Fluorescent E7-A42K85R, A87T, E90P Poorly Fluorescent

[0204] b. Effect of Phosphorylation on the Fluorescence Changes in GFPAfter Quench.

[0205] Analysis of the effect of quenching on the fluorescence of themutants Erk-6, Erk-7 and Erk-6-B 17 was performed. The results presentedin Table XII demonstrate improved fluorescent changes in response toquenching with acetate buffer. These results demonstrate that themutagenesis approaches are generally applicable to improve fluorescenceand phosphorylation dependent changes in quenching.

[0206] Table XII: Effects of quenching on the fluorescence of Erk-6 andErk-7 mutants TABLE XII Effects of quenching on the fluorescence ofErk-6 and Erk-7 mutants Fold Increase in Fluorescence Constructs afterPhosphorylation Erk-6 1.20 Erk-6-17B 1.40 Erk-7 1.05

[0207] These experiments were performed by incubating the GFP sample (2μM) with Erk-1 kinase (1 μg) for 1 hour at 30° C. in assay buffer (20 mMMOPS. pH 7.2, 25 mM beta-glycoerol phosphate, 5 mM EGTA. 1 mM DTT, 1 mMATP, 20 mM MgCl₂). Reactions were quenched by the addition of acetatequenching buffer (100 μL of 100 mM Acetate pH 5.0, 100 mM NaCl, 20 mMbeta-Glycerol phosphate). Fluorescence changes were measured after 3hours of incubation in acetate quenching buffer. Results represent themeans of triplicate determinations.

[0208] The mutant Erk-6-B17 exhibited 1.4 times greater fluorescencethan the original construct Erk-6. Incubation of this mutant with anexcess activated kinase resulted in larger change in fluorescence afterquenching. These results demonstrate that these methods of improvingmutants are generally applicable to the creation and improvement of arange of phosphorylation motifs.

[0209] F. PKC Selective Phosphorylation Motifs

[0210] The rates of phosphorylation of GFP having PKC motifs weredetermined by measuring ³²P incorporation in the presence of differentPKC isoforms (Table XIII). These examples include one example where amembrane association motif is part of the GFP mutant. TABLE XIII Rate ofPhosphorylation of Various GFP Kinase Substrates Using Various KinasesKinase and Activity (CPM) GFP Mutant K8 Having the IndicatedPhosphorylation Motif PKC alpha PKC ε PKC ζ PKC alpha 10,629 21,7348,129 PKC alpha + Membrane 22,675 10,230 2,138 Association motif(Hepta-Lys) PKC β1 13,332 39,533 20,173 PKC δ 4,935 12,473 7,733 PKC ε11,310 43,705 26,783 PKC ζ 4,745 12,688 8,421 PKC μ 5,230 20,259 14,606

[0211] These experiments were performed by incubating the indicated GFPmutant (5 micromolar) with the indicated PKC isoform (0.2 μg) for 30minutes at 30° C. in 25 mM TRIS pH 7.5. 1 mM DTT, 10 mM MgCl₂ 0.1 mM ATP20 μg/ml phosphatidylserine, 10 μM OAG, 200 μM CaCl₂ (for PKCα) and 1 mMEGTA (for PKCs ε and ζ). These results demonstrate that the availablesequence diversity available at the N-terminus of GFP is sufficient togenerate isoform specific phosphorylation of different mutants. Therelative specificities identified for the GFP substrates in thisexperiment broadly matched those identified by Nishikawa et al (1997)who used non-GFP peptides to selectively measure PCK isoform activity.GFP contains an endogenous phosphorylation site (underlined) (Gly HisLys Phe Ser Val Ser Gly) within a relatively poorly recognizedphosphorylation recognition motif that may be phosphorylated by some PKCisoforms. This may reduce the apparent specificity of the N-terminalphosphorylation motifs as measured by ³²P-incorporation because thesedata represent phosphorylation both at the N-terminal site and theinternal site. Membrane association motif poly-Lys (hepta-Lys) was addedto the C-terminus of GFP mutant K8 with PKC alpha phosphorylation motifat the N-terminus.

[0212] 1. Determination of Fluorescence Changes in Response toPhosphorylation

[0213] To determine if changes in fluorescence correlated with changesin phoshorylation, the previous experiments were repeated except thatthe fluorescence changes rather than ³²P incorporation were measuredafter the addition of a quenching agent. These results demonstrate thatfluorescence changes in the GFP samples after quenching correlate withthe incorporation of ³²P (see Table XIV).

[0214] Maximal quenching for the PKC substrates was observed with 50 mMacetate buffer at pH 5.2 in the presence of 20% DMSO. The maximum changein fluorescence observed was typically 1.6 to 2.0 fold greaterfluorescence for the phosphorylated substrate after 24 hours under thebest conditions identified. The difference in quench conditions for thecase of the PKC specific substrates compared to the PKA substrate may bedue to the large hydrophobic motif C-terminal to the site ofphosphorylation in these substrates (Ser-Phe-(Phe/Arg)-Phe).

[0215] 2. Addition of Membrane Association and Protein-ProteinInteraction Motifs to GFP

[0216] A polybasic membrane association motif derived from K-ras(Hancock et al. EMBO J. 12:4033-4039 (1991))) (Hepta-Lys) was added tothe C-terminus of the PKC-alpha GFP by PCR. In addition, the farnesylmodification site could be added to the Hepta-Lys motif, resulting inthe sequence Lys-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Lys-Thr-Lys-Cys-Val-Ile-Met(SEQ ID NO:63) to create a tighter membrane association. Thephosphorylation kinetics of this GFP was compared to that of a GFP thatcontained the PKC-alpha specific phosphorylation motif, but not themembrane associated motif. Both the constructs were highly fluorescentand were expressed at high levels in bacteria. The putative membraneassociated GFP was soluble in aqueous solution at high saltconcentration (0.3 M NaCl), but precipitated upon storage after dialysisto 0.1 M NaCl. All experiments using this protein were conducted onmaterial that was stored in high salt and diluted into low ionicstrength media in the presence of phospholipid vesicles immediatelyprior to experiments.

[0217] The addition of a membrane association motif significantlyincreased the rate of phosphorylation of the substrate compared to a GFPsubstrate with the same phosphorylation recognition motif, but lackingthe membrane association motif (FIG. 9). The addition of the membranerecognition motif also had a significant effect on the specificity ofthe PKC alpha with respect to other PKC isoforms (Table XIII). Kineticanalysis of the PKC alpha substrates with or without the membraneassociation motif reveals that increased phosphorylation was primarilydue to an increase in apparent Km of the substrate, with little effecton the Vmax (FIG. 9).

[0218] All publications and patent documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication or patent document were soindividually denoted.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 74 <210> SEQ ID NO 1<211> LENGTH: 716 <212> TYPE: DNA <213> ORGANISM: Aequorea <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(714) <400> SEQUENCE:1 atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt 48 MetSer Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15gaa tta gat ggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag 96 GluLeu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggtgaa ggt gat gca aca tac gga aaa ctt acc ctt aaa ttt att tgc 144 Gly GluGly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 act actgga aaa cta cct gtt cca tgg cca aca ctt gtc act act ttc 192 Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggtgtt caa tgc ttt tca aga tac cca gat cat atg aaa cgg 240 Ser Tyr Gly ValGln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 cat gac tttttc aag agt gcc atg ccc gaa ggt tat gta cag gaa aga 288 His Asp Phe PheLys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt ttcaaa gat gac ggg aac tac aag aca cgt gct gaa gtc 336 Thr Ile Phe Phe LysAsp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggtgat acc ctt gtt aat aga atc gag tta aaa ggt att 384 Lys Phe Glu Gly AspThr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaagat gga aac att ctt gga cac aaa ttg gaa tac aac 432 Asp Phe Lys Glu AspGly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tca cacaat gta tac atc atg gca gac aaa caa aag aat gga 480 Tyr Asn Ser His AsnVal Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 atc aaa gttaac ttc aaa att aga cac aac att gaa gat gga agc gtt 528 Ile Lys Val AsnPhe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 caa cta gcagac cat tat caa caa aat act cca att ggc gat ggc cct 576 Gln Leu Ala AspHis Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt ttacca gac aac cat tac ctg tcc aca caa tct gcc ctt tcg 624 Val Leu Leu ProAsp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat cccaac gaa aag aga gac cac atg gtc ctt ctt gag ttt gta 672 Lys Asp Pro AsnGlu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 aca gct gctggg att aca cat ggc atg gat gaa cta tac aaa ta 716 Thr Ala Ala Gly IleThr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> SEQ ID NO 2 <211>LENGTH: 238 <212> TYPE: PRT <213> ORGANISM: Aequorea <400> SEQUENCE: 2Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 1015 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 2530 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 4045 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 5560 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 7075 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 8590 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys GlyIle 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu GluTyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys GlnLys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn IleGlu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn ThrPro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr LeuSer Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg AspHis Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr HisGly Met Asp Glu Leu Tyr Lys 225 230 235 <210> SEQ ID NO 3 <211> LENGTH:5 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: phosphorylation recognition motif for proteinkinase A <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)..(5)<223> OTHER INFORMATION: Xaa at residue 3 is any amino acid Xaa atresidue 5 is a hydrophobic amino acid <400> SEQUENCE: 3 Arg Arg Xaa SerXaa 1 5 <210> SEQ ID NO 4 <211> LENGTH: 5 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:phosphorylation recognition motif for protein kinase A <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (1)..(5) <223> OTHERINFORMATION: Xaa at residue 3 is any amino acid Xaa at residue 5 is ahydrophobic amino acid <400> SEQUENCE: 4 Arg Arg Xaa Thr Xaa 1 5 <210>SEQ ID NO 5 <211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: cGMP-dependent proteinkinase phosphorylation recognition motif <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: (1)..(13) <223> OTHER INFORMATION: Xaa is eitherlysine or arginine, and the first S is the site of phosphorylation <400>SEQUENCE: 5 Xaa Lys Ile Ser Ala Ser Glu Phe Asp Arg Pro Leu Arg 1 5 10<210> SEQ ID NO 6 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: residues forthe cGMP-dependent protein kinase phosphorylation recognition motif<400> SEQUENCE: 6 Asp Arg Pro Leu Arg 1 5 <210> SEQ ID NO 7 <211>LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: synthetic substrates for proteinkinase C <220> FEATURE: <221> NAME/KEY: variant <222> LOCATION: (1)..(8)<223> OTHER INFORMATION: Xaa is any amino acid <400> SEQUENCE: 7 Xaa ArgXaa Xaa Ser Xaa Arg Xaa 1 5 <210> SEQ ID NO 8 <211> LENGTH: 9 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: phosphorylation recognition motif <400> SEQUENCE: 8 Lys LysLys Lys Arg Phe Ser Phe Lys 1 5 <210> SEQ ID NO 9 <211> LENGTH: 9 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: phosphorylation recognition motif <400> SEQUENCE: 9 Leu ArgArg Leu Ser Asp Ser Asn Phe 1 5 <210> SEQ ID NO 10 <211> LENGTH: 10<212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: recognition sequence around the phosphorylation site<400> SEQUENCE: 10 Lys Lys Leu Asn Arg Thr Leu Thr Val Ala 1 5 10 <210>SEQ ID NO 11 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: recognition sequencearound the phosphorylation site <400> SEQUENCE: 11 Lys Lys Ala Asn ArgThr Leu Ser Val Ala 1 5 10 <210> SEQ ID NO 12 <211> LENGTH: 10 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Substrate <400> SEQUENCE: 12 Met Arg Arg Arg Arg Ser IleIle Thr Gly 1 5 10 <210> SEQ ID NO 13 <211> LENGTH: 13 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: substrate <400> SEQUENCE: 13 Met Arg Arg Arg Arg Ser IleIle Ile Ile Phe Thr Gly 1 5 10 <210> SEQ ID NO 14 <211> LENGTH: 5 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: phosphorylation recognition motif <400> SEQUENCE: 14 ArgArg Phe Ser Ala 1 5 <210> SEQ ID NO 15 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: phosphorylation recognition motif <400> SEQUENCE: 15 LysArg Asp Ser Met 1 5 <210> SEQ ID NO 16 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: phosphorylation motif <400> SEQUENCE: 16 Met Ser Lys ArgArg Asp Ser Leu Thr 1 5 <210> SEQ ID NO 17 <211> LENGTH: 3 <212> TYPE:DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: randomly generated mixtures of oligonucleotides <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(3) <223>OTHER INFORMATION: n is A, C, G, or T, and k is G or T An exemplifiedcodon motiff (NNK)6 <400> SEQUENCE: 17 nnk 3 <210> SEQ ID NO 18 <211>LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: mutant <400> SEQUENCE: 18 Arg Arg LeuSer Ile 1 5 <210> SEQ ID NO 19 <211> LENGTH: 5 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:change in fluorescence for mutants <400> SEQUENCE: 19 Arg Arg Phe SerVal 1 5 <210> SEQ ID NO 20 <211> LENGTH: 5 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:change in fluorescence for mutants <400> SEQUENCE: 20 Arg Arg Phe SerArg 1 5 <210> SEQ ID NO 21 <211> LENGTH: 5 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:change in fluorescence for mutants <400> SEQUENCE: 21 Arg Arg Ser IlePhe 1 5 <210> SEQ ID NO 22 <211> LENGTH: 6 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:change in fluorescence for mutants <400> SEQUENCE: 22 Arg Arg Gly SerIle Leu 1 5 <210> SEQ ID NO 23 <211> LENGTH: 6 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:change in fluorescence for mutants <400> SEQUENCE: 23 Lys Arg Lys SerGly Ile 1 5 <210> SEQ ID NO 24 <211> LENGTH: 5 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:change in fluorescence for mutants <400> SEQUENCE: 24 Arg Arg Gly SerVal 1 5 <210> SEQ ID NO 25 <211> LENGTH: 8 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:phosphorylation site inserted at the N-terminus <400> SEQUENCE: 25 MetArg Lys Gly Ser Cys Leu Phe 1 5 <210> SEQ ID NO 26 <211> LENGTH: 8 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: phosphorylation site inserted at the N-terminus <400>SEQUENCE: 26 Met Arg Lys Gly Ser Leu Leu Phe 1 5 <210> SEQ ID NO 27<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: phosphorylation site inserted atthe N-terminus <400> SEQUENCE: 27 Met Arg Arg Glu Ser Leu Leu Phe 1 5<210> SEQ ID NO 28 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:phosphorylation site inserted at the N-terminus <400> SEQUENCE: 28 MetArg Asp Ser Cys Leu Phe 1 5 <210> SEQ ID NO 29 <211> LENGTH: 8 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: phosphorylation site inserted at the N-terminus <400>SEQUENCE: 29 Met Ser Arg Arg Asp Ser Cys Phe 1 5 <210> SEQ ID NO 30<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: phosphorylation site inserted atthe N-terminus <400> SEQUENCE: 30 Met Ser Lys Arg Arg Asp Ser Leu 1 5<210> SEQ ID NO 31 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:phosphorylation site inserted at the N-terminus <400> SEQUENCE: 31 MetSer Arg Arg Arg Ser Ile 1 5 <210> SEQ ID NO 32 <211> LENGTH: 8 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: phosphorylation site inserted at the N-terminus <400>SEQUENCE: 32 Met Arg Arg Arg Arg Ser Ile Ile 1 5 <210> SEQ ID NO 33<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: phosphorylation site inserted atthe N-terminus <400> SEQUENCE: 33 Met Arg Arg Arg Arg Ser Ile Ile Ile 15 <210> SEQ ID NO 34 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:phosphorylation site inserted at the N-terminus <400> SEQUENCE: 34 MetArg Arg Arg Arg Ser Ile Ile Ile Phe 1 5 10 <210> SEQ ID NO 35 <211>LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: phosphorylation site inserted at theN-terminus <400> SEQUENCE: 35 Met Arg Arg Arg Arg Ser Ile Ile Ile IlePhe 1 5 10 <210> SEQ ID NO 36 <211> LENGTH: 8 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:phosphorylation motif <400> SEQUENCE: 36 Met Ser Lys Gly Glu Glu Leu Phe1 5 <210> SEQ ID NO 37 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: HIV-1protease <400> SEQUENCE: 37 Ser Gln Asn Tyr Pro Ile Val Gln 1 5 <210>SEQ ID NO 38 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: HIV-1 protease <400>SEQUENCE: 38 Lys Ala Arg Val Leu Ala Glu Met Ser 1 5 <210> SEQ ID NO 39<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: prohormone convertase <400>SEQUENCE: 39 Pro Ser Pro Arg Glu Gly Lys Arg Ser Tyr 1 5 10 <210> SEQ IDNO 40 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION:interleukin-1b-converting enzyme <400> SEQUENCE: 40 Tyr Val Ala Asp Gly1 5 <210> SEQ ID NO 41 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Adenovirusendopeptidase <400> SEQUENCE: 41 Met Phe Gly Gly Ala Lys Lys Arg 1 5<210> SEQ ID NO 42 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:cytomegalovirus assemblin <400> SEQUENCE: 42 Gly Val Val Met Ala Ser SerArg Leu Ala 1 5 10 <210> SEQ ID NO 43 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Leishmanolysin <400> SEQUENCE: 43 Leu Ile Ala Tyr Ile LeuLys Lys Ala Thr 1 5 10 <210> SEQ ID NO 44 <211> LENGTH: 7 <212> TYPE:PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: b-Secretase for amyloid precursor protein <400> SEQUENCE:44 Val Lys Met Asp Ala Glu Phe 1 5 <210> SEQ ID NO 45 <211> LENGTH: 17<212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: thrombin <400> SEQUENCE: 45 Phe Leu Ala Glu Gly GlyGly Val Arg Gly Pro Arg Val Val Glu Arg 1 5 10 15 His <210> SEQ ID NO 46<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: renin andangiotension-converting enzyme <400> SEQUENCE: 46 Asp Arg Val Tyr IleHis Pro Phe His Leu Val Ile His 1 5 10 <210> SEQ ID NO 47 <211> LENGTH:8 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: cathepsin D <400> SEQUENCE: 47 Lys Pro Ala LeuPhe Phe Arg Leu 1 5 <210> SEQ ID NO 48 <211> LENGTH: 30 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Kininogenases including kallikrein <400> SEQUENCE: 48 GlnPro Leu Gly Gln Thr Ser Leu Met Lys Arg Pro Pro Gly Phe Ser 1 5 10 15Pro Phe Arg Ser Val Gln Val Met Lys Thr Gln Glu Gly Ser 20 25 30 <210>SEQ ID NO 49 <211> LENGTH: 240 <212> TYPE: PRT <213> ORGANISM: GFPmutant K4 <400> SEQUENCE: 49 Met Gly Arg Arg Arg Ala Ser Ile Ile Phe ThrGly Val Val Pro Ile 1 5 10 15 Leu Val Glu Leu Asp Gly Asp Val Asn GlyHis Lys Phe Ser Val Ser 20 25 30 Gly Glu Gly Glu Gly Asp Ala Thr Tyr GlyLys Leu Thr Leu Lys Phe 35 40 45 Ile Cys Thr Thr Gly Lys Leu Pro Val ProTrp Pro Thr Leu Val Thr 50 55 60 Thr Phe Ala Tyr Gly Val Gln Cys Phe SerArg Tyr Pro Asp His Met 65 70 75 80 Lys Arg His Asp Phe Phe Lys Ser AlaMet Pro Glu Gly Tyr Val Gln 85 90 95 Glu Arg Thr Ile Phe Phe Lys Asp AspGly Asn Tyr Lys Thr Arg Ala 100 105 110 Glu Val Lys Phe Glu Gly Asp ThrLeu Val Asn Arg Ile Glu Leu Lys 115 120 125 Gly Ile Asp Phe Lys Glu AspGly Asn Ile Leu Gly His Lys Leu Glu 130 135 140 Tyr Asn Tyr Asn Ser HisLys Val Tyr Ile Met Ala Asp Lys Gln Lys 145 150 155 160 Asn Gly Ile LysAla Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly 165 170 175 Ser Val GlnLeu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp 180 185 190 Gly ProVal Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala 195 200 205 LeuSer Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu 210 215 220Phe Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230235 240 <210> SEQ ID NO 50 <211> LENGTH: 9 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:Cyclin B-CDC2 <400> SEQUENCE: 50 His His His Lys Ser Pro Arg Arg Arg 1 5<210> SEQ ID NO 51 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: cyclinA-CDK2 <400> SEQUENCE: 51 His His His Arg Ser Arg Pro Lys Arg 1 5 <210>SEQ ID NO 52 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificialsequence <220> FEATURE: <223> OTHER INFORMATION: protein kinase A <400>SEQUENCE: 52 Arg Arg Arg Arg Ser Ile Ile Phe Ile 1 5 <210> SEQ ID NO 53<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial sequence<220> FEATURE: <223> OTHER INFORMATION: SLK 1 <400> SEQUENCE: 53 Arg ArgPhe Gly Ser Leu Arg Arg Leu 1 5 <210> SEQ ID NO 54 <211> LENGTH: 9 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: ERK 1 <400> SEQUENCE: 54 Thr Gly Pro Leu Ser Pro Gly ProPhe 1 5 <210> SEQ ID NO 55 <211> LENGTH: 13 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:protein kinase C alpha <400> SEQUENCE: 55 Arg Arg Arg Arg Arg Lys GlySer Phe Arg Arg Lys Ala 1 5 10 <210> SEQ ID NO 56 <211> LENGTH: 10 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Phosphorylation motif <400> SEQUENCE: 56 Arg Arg Arg ArgArg Lys Gly Ser Phe Arg 1 5 10 <210> SEQ ID NO 57 <211> LENGTH: 10 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Phosphorylation motif <400> SEQUENCE: 57 Arg Arg Arg ArgArg Lys Gly Ser Phe Arg 1 5 10 <210> SEQ ID NO 58 <211> LENGTH: 10 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Phosphorylation motif <400> SEQUENCE: 58 Phe Lys Leu LysArg Lys Gly Ser Phe Lys 1 5 10 <210> SEQ ID NO 59 <211> LENGTH: 10 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Phosphorylation motif <400> SEQUENCE: 59 Ala Arg Arg LysArg Lys Gly Ser Phe Phe 1 5 10 <210> SEQ ID NO 60 <211> LENGTH: 10 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Phosphorylation motif <400> SEQUENCE: 60 Tyr Tyr Ala LysArg Lys Met Ser Phe Phe 1 5 10 <210> SEQ ID NO 61 <211> LENGTH: 10 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Phosphorylation motif <400> SEQUENCE: 61 Arg Arg Phe LysArg Gln Gly Ser Phe Phe 1 5 10 <210> SEQ ID NO 62 <211> LENGTH: 10 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: Phosphorylation motif <400> SEQUENCE: 62 Ala Ala Leu ValArg Gln Met Ser Val Ala 1 5 10 <210> SEQ ID NO 63 <211> LENGTH: 15 <212>TYPE: PRT <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: hepta-lys motif <400> SEQUENCE: 63 Lys Lys Lys Lys Lys LysLys Ser Lys Thr Lys Cys Val Ile Met 1 5 10 15 <210> SEQ ID NO 64 <211>LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Phosphorylation motif <400> SEQUENCE:64 Met Val Glu Pro Leu Thr Pro Ser Phe 1 5 <210> SEQ ID NO 65 <211>LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Phosphorylation motif <400> SEQUENCE:65 Met Thr Gly Pro Leu Ser Pro Gly Phe 1 5 <210> SEQ ID NO 66 <211>LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Phosphorylation motif <400> SEQUENCE:66 Met Thr Gly Pro Leu Ser Pro Gly Tyr 1 5 <210> SEQ ID NO 67 <211>LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Phosphorylation motif <400> SEQUENCE:67 Met Thr Gly Pro Leu Ser Pro Gly Leu 1 5 <210> SEQ ID NO 68 <211>LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: Phosphorylation motif <400> SEQUENCE:68 Met Thr Gly Pro Leu Ser Pro Gly Pro 1 5 <210> SEQ ID NO 69 <211>LENGTH: 190 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220>FEATURE: <223> OTHER INFORMATION: FRAGMENT OF pRSET B VECTOR <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (13)..(135) <400> SEQUENCE:69 ggagatatac at atg cgg ggt tct cat cat cat cat cat cat ggt atg gct 51Met Arg Gly Ser His His His His His His Gly Met Ala 1 5 10 agc atg actggt gga cag caa atg ggt cgg gat ctg tac gac gat gac 99 Ser Met Thr GlyGly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp 15 20 25 gat aag gat cccccc gct gaa ttc atg agt tac aaa taataaggat 145 Asp Lys Asp Pro Pro AlaGlu Phe Met Ser Tyr Lys 30 35 40 ccgagctcga gatctgcagc tggtaccatggaattcgaag gttga 190 <210> SEQ ID NO 70 <211> LENGTH: 41 <212> TYPE: PRT<213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHERINFORMATION: FRAGMENT OF pRSET B VECTOR <400> SEQUENCE: 70 Met Arg GlySer His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly GlyGln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro ProAla Glu Phe Met Ser Tyr Lys 35 40 <210> SEQ ID NO 71 <211> LENGTH: 167<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223>OTHER INFORMATION: FRAGMENT OF pRSET B VECTOR <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (13)..(69) <400> SEQUENCE: 71 ggagatatacat atg cgg ggt tct cat cat cat cat cat cat ggt atg gct 51 Met Arg GlySer His His His His His His Gly Met Ala 1 5 10 agc atg act ggt gga cagcaaatgggtc gggatctgta cgacgatgac 99 Ser Met Thr Gly Gly Gln 15gataaggatc cgagctcgag atctgcagct ggtaccatga gaagaagaag atcaaaataa 159aagcttga 167 <210> SEQ ID NO 72 <211> LENGTH: 19 <212> TYPE: PRT <213>ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION:FRAGMENT OF pRSET B VECTOR <400> SEQUENCE: 72 Met Arg Gly Ser His HisHis His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln <210> SEQID NO 73 <211> LENGTH: 717 <212> TYPE: DNA <213> ORGANISM: Aequoreagreen fluorescent protein phosphorylation mutant <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(714) <400> SEQUENCE: 73 atg agt aaagga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt 48 Met Ser Lys GlyGlu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 gaa tta gatggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag 96 Glu Leu Asp GlyAsp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gaa ggt gatgca aca tac gga aaa ctt acc ctt aaa ttt att tgc 144 Gly Glu Gly Asp AlaThr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 act act gga aaa ctacct gtt cca tgg cca aca ctt gtc act act ttc 192 Thr Thr Gly Lys Leu ProVal Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggt gtt caa tgcttt tca aga tac cca gat cat atg aaa cag 240 Ser Tyr Gly Val Gln Cys PheSer Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 cat gac ttt ttc aag agtgcc atg ccc gaa ggt tat gta cag gaa aga 288 His Asp Phe Phe Lys Ser AlaMet Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 tct ata ttt ttc aaa gat gacggg aac tac aag aca cgt gct gaa gtc 336 Ser Ile Phe Phe Lys Asp Asp GlyAsn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggt gat acc cttgtt aat aga atc gag tta aaa ggt att 384 Lys Phe Glu Gly Asp Thr Leu ValAsn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaa gat gga aacatt ctt gga cac aaa ttg gaa tac aac 432 Asp Phe Lys Glu Asp Gly Asn IleLeu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tca cac aat gta tacatc atg gca gac aaa caa aag aat gga 480 Tyr Asn Ser His Asn Val Tyr IleMet Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 atc aaa gtt aac ttc aaaatt aga cac aac att gaa gat gga agc gtt 528 Ile Lys Val Asn Phe Lys IleArg His Asn Ile Glu Asp Gly Ser Val 165 170 175 caa cta gca gac cat tatcaa caa aat act cca att ggc gat ggc cct 576 Gln Leu Ala Asp His Tyr GlnGln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt tta cca gac aaccat tac ctg tcc aca caa tct gcc ctt tcg 624 Val Leu Leu Pro Asp Asn HisTyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat ccc aac gaa aagaga gac cac atg gtc ctt ctt gag ttt gta 672 Lys Asp Pro Asn Glu Lys ArgAsp His Met Val Leu Leu Glu Phe Val 210 215 220 aca gct gct ggg att acacat ggc atg gat gaa cta tac aaa taa 717 Thr Ala Ala Gly Ile Thr His GlyMet Asp Glu Leu Tyr Lys 225 230 235 <210> SEQ ID NO 74 <211> LENGTH: 238<212> TYPE: PRT <213> ORGANISM: Aequorea green fluorescent proteinphosphorylation mutant <400> SEQUENCE: 74 Met Ser Lys Gly Glu Glu LeuPhe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp ValAsn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala ThrTyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu ProVal Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ser Tyr Gly Val Gln CysPhe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 His Asp Phe Phe LysSer Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe LysAsp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu GlyAsp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe LysGlu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr AsnSer His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu PheVal 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys225 230 235

I claim:
 1. A fluorescent compound for detecting an activity,comprising: a fluorescent protein moiety, and at least one exogenoussubstrate recognition motif for an activity, wherein said fluorescentprotein moiety can be converted from a first state to a second state inresponse to said activity, further wherein said fluorescent compoundexhibits at least one different fluorescent property in said first stateand said second state under quenching conditions.
 2. The fluorescentcompound of claim 1, wherein said activity is an enzymatic activity. 3.The fluorescent compound of claim 2, wherein said enzymatic activity isselected from the group consisting of a kinase activity, a phosphataseactivity, a protease activity, a glycosylation activity, and a farnesyltransferase activity.
 4. The fluorescent compound of claim 1, whereinsaid fluorescent protein moiety comprises an Aequorea-relatedfluorescent protein.
 5. The fluorescent compound of claim 1, whereinsaid fluorescent protein moiety comprises a phosphorylation recognitionmotif for a protein kinase is a serine/threonine specific proteinkinase.
 6. The fluorescent compound of claim 1, wherein said fluorescentprotein moiety comprises a phosphorylation recognition motif for aprotein kinase selected from the group consisting of protein kinase A, acGMP-dependent protein kinase, protein kinase C,Ca²⁺/calmodulin-dependent protein kinase I, Ca²⁺/calmodulin-dependentprotein kinase II, and MAP kinase activated protein kinase.
 7. Thefluorescent compound of claim 4, wherein said Aequorea-relatedfluorescent protein moiety comprises the mutations in GFP mutant K8. 8.The fluorescent compound of claim 4, wherein said at least one exogenoussubstrate recognition motif for an activity is within the first 20 aminoacids of the amino terminus of said Aequorea-related fluorescent proteinmoiety.
 9. The fluorescent compound of claim 4, wherein said at leastone exogenous substrate recognition motif for an enzymatic activity iswithin the first 10 amino acids of the amino terminus of saidAequorea-related fluorescent protein moiety.
 10. The fluorescentcompound of claim 1, wherein said quenching conditions is acidquenching.
 11. The fluorescent compound of claim 4, wherein saidAequorea-related fluorescent protein moiety is membrane associated. 12.The fluorescent compound of claim 11, wherein said Aequorea-relatedfluorescent moiety comprises a poly-Lys region.
 13. The fluorescentcompound of claim 4, wherein said Aequorea-related fluorescent proteinmoiety comprises a protein-protein interaction domain.
 14. Thefluorescent compound of claim 4, wherein said Aequorea-relatedfluorescent moiety is membrane bound.
 15. A nucleic acid molecule codingfor the expression of a fluorescent compound, wherein said fluorescentcompound comprises a fluorescent protein moiety, and at least oneexogenous substrate motif for an activity, further wherein saidfluorescent protein moiety can be converted from a first state to asecond state by an activity, further wherein said first state and saidsecond state can be differentiated under quenching conditions.
 16. Acell, comprising: a nucleic acid molecule coding for the expression of afluorescent compound, wherein said fluorescent compound comprises afluorescent protein moiety, and at least one exogenous substrate motiffor an activity, wherein said fluorescent protein moiety can beconverted from a first state to a second state by an activity, furtherwherein said first state and said second state can be differentiatedunder quenching conditions.
 17. A method for determining whether asample contains an activity, comprising: contacting a sample with afluorescent compound, wherein said fluorescent compound comprises afluorescent protein moiety, and at least one exogenous substrate motiffor an activity, further wherein said fluorescent protein moiety can beconverted from a first state to a second state by an activity, excitingsaid fluorescent compound, and measuring the amount of emission fromsaid fluorescent compound.
 18. The method of claim 17, furthercomprising the step of comparing the amount of emission measured fromsaid fluorescent compound with the emission from a control sample. 19.The method of claim 18, wherein said enzymatic activity is selected fromthe group consisting of a kinase activity, a phosphatase activity, aprotease activity, a glycosylase activity, and a farnsyl transferaseactivity.
 20. The method of claim 19, wherein said quenching is acidquenching.
 21. A method for determining whether a cell exhibits anactivity comprising the steps of: exciting a transfected host cellcomprising a recombinant nucleic acid molecule, wherein said recombinantnucleic acid molecule comprises at least one expression control sequenceoperatively linked to a nucleic acid sequence coding for the expressionof a fluorescent compound, wherein said fluorescent compound comprises afluorescent protein moiety, and a substrate motif for an activity,further wherein said fluorescent compound can be converted from a firststate to a second state in the presence of said activity, furtherwherein said first state and said second state and be differentiatedunder quenching conditions, and measuring the emission from saidfluorescent compound.
 22. The method of claim 21, wherein said enzymaticactivity is selected from the group consisting of a kinase activity, aphosphatase activity, a protease activity, a glycosylase activity, and afarnsyl transferase activity.
 23. A method for determining whether asample contains an enzymatic activity, comprising: a) contacting asample with a fluorescent compound, wherein said fluorescent compoundcomprises an Aequorea-related fluorescent protein moiety and anexogenous substrate motif for an enzymatic activity, further whereinsaid fluorescent compound can be converted from a first state to asecond state by said enzymatic activity, further wherein said firststate and said second state can be differentiated under quenchingconditions, b) exciting said fluorescent compound, and c) measuring theamount of fluorescence emitted from said sample, whereby the amount ofquenching that is consistent with the presence of said enzyme activityindicates the presence of said enzyme activity in said sample.
 24. Acompound identified by the method comprising the steps of: a) contactinga sample with a fluorescent compound, wherein said fluorescent compoundcomprises a fluorescent protein moiety and an exogenous substrate motiffor an activity, further wherein said fluorescent compound can beconverted from a first state to a second state by said activity, furtherwherein said first state and said second state can be differentiatedunder quenching conditions, b) exciting said fluorescent compound, andc) measuring the amount of fluorescence emitted from said sample.